Amplification method and primers for use therein

Abstract

To provide an improved method of amplifying a nucleic acid region of interest, and primers for use therein.SOLUTION: The present invention provides an improved method of amplifying a nucleic acid region of interest that has resulted from the recombination of two or more immunoglobulin or T cell receptor gene segments, and primers for use therein, where the method includes performing an amplification step at an annealing temperature determined relative to the critical annealing temperature unique to the reaction and / or using optimised primers, thereby amplifying recombined immunological or T cell receptor genes.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention generally relates to improved methods for amplifying a nucleic acid region of interest, and the primers used therein. More particularly, the present invention relates to a method for the preparation of a fusion protein comprising the steps of: Improved methods for amplifying nucleic acid regions resulting from rearrangements of receptor gene segments; and The method of the present invention relates to a primer set specific to a given reaction. and / or performing an amplification step at an annealing temperature determined relative to the amplification temperature. Using optimized primers, it is possible to amplify immunological or T cell receptor gene rearrangements. This allows for a higher level of sensitivity than previously achievable with respect to prior art methods. The method of the present invention is particularly useful when the subject rearrangement target includes a unique N region. It is a sensitive yet simple method to detect specific immunological and T cell receptor nucleic acid rearrangement events. Providing a means to identify specific V / D / J rearrangement events (e.g., minimal residual disease in leukemia) detection) or characterization of immunological or T cell receptor gene regions of interest. including, but not limited to, diagnosing and / or monitoring clonal lymphoid cell populations or disease states. It is useful for a wide range of applications, including [Background technology]

[0002] Any prior publications (or information derived therefrom) or known Reference to any matter disclosed herein is to its prior publication (or information derived therefrom) or to any that the matter of knowledge forms part of the general common knowledge in the field of the present application; It is not and should not be taken as an endorsement or condonation of any kind.

[0003] The bibliographic details of the publications referred to by the author in this specification are compiled alphabetically at the end of the description.

[0004] Clones are generally understood as a population of cells derived from a common progenitor cell. Diagnosing and / or detecting the presence of a clone cell population or organism in a subject generally constitutes a relatively problematic procedure. Specifically, a clone population may constitute only a small component within a larger population of cells or organisms. For example, with respect to mammalian organisms, one of the more common situations where the detection of a clone cell population is required arises in the diagnosis and / or detection of neoplasms such as cancer. However, the detection of one or more clone populations can also be important in the diagnosis of conditions such as myelodysplasia or polycythemia vera, and also in the detection of antigen-driven clones generated by the immune system.

[0005] Generally, the population in which a clone occurs corresponds to a population of cells within a specific tissue or compartment of the body. Nevertheless, sampling such a cell population, despite effectively narrowing the examination to a subpopulation of cells or organisms, can present to the clinician within the large background population of non-clone cells or organisms in which the clone population must be identified.

[0006] When the members of a clone are characterized by a molecular marker such as a change in the DNA sequence, the detection problem may in some cases be convertible to the problem of detecting a molecular population having the same molecular sequence within a large molecular population having different sequences, all of which are similar and different, or ​​​​​​​​​​​​​​or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules. Certain classes of molecular markers are very specific but present unique complexities with regard to detection, which result from gene rearrangement events. The rearrangement of genetic material in somatic cells involves bringing together two or more initially separated regions of the genome. It can occur as a random process, but it can also occur as part of the development process of normal lymphoid cells. In relation to cancer, the rearrangement can be simple or complex. A simple rearrangement may be considered one in which two unrelated genes or regions are juxtaposed. A complex rearrangement can be considered one in which three or more genes or gene segments are rearranged. A typical example of a complex rearrangement occurs during the normal development of lymphoid cells and involves the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) variable genes with the rearrangement of V, D, and J gene segments. The loci of these gene segments are widely separated in the germline, but the rearrangement during lymphocyte development results in the juxtaposition of V, D, and J gene segments, or V and J gene segments, and the junctions between these gene segments are characterized by small regions of nucleotide insertions and deletions (N1 and N2 regions). This process occurs randomly.

[0007] or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules. Certain classes of molecular markers are very specific but present unique complexities with regard to detection, which result from gene rearrangement events.

[0008] The rearrangement of genetic material in somatic cells involves bringing together two or more initially separated regions of the genome. It can occur as a random process, but it can also occur as part of the development process of normal lymphoid cells. In relation to cancer, the rearrangement can be simple or complex. A simple rearrangement may be considered one in which two unrelated genes or regions are juxtaposed. A complex rearrangement can be considered one in which three or more genes or gene segments are rearranged. A typical example of a complex rearrangement occurs during the normal development of lymphoid cells and involves the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) variable genes with the rearrangement of V, D, and J gene segments. The loci of these gene segments are widely separated in the germline, but the rearrangement during lymphocyte development results in the juxtaposition of V, D, and J gene segments, or V and J gene segments, and the junctions between these gene segments are characterized by small regions of nucleotide insertions and deletions (N1 and N2 regions). This process occurs randomly. or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules.

[0009] or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules. Certain classes of molecular markers are very specific but present unique complexities with regard to detection, which result from gene rearrangement events. The rearrangement of genetic material in somatic cells involves bringing together two or more initially separated regions of the genome. It can occur as a random process, but it can also occur as part of the development process of normal lymphoid cells. In relation to cancer, the rearrangement can be simple or complex. A simple rearrangement may be considered one in which two unrelated genes or regions are juxtaposed. A complex rearrangement can be considered one in which three or more genes or gene segments are rearranged. A typical example of a complex rearrangement occurs during the normal development of lymphoid cells and involves the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) variable genes with the rearrangement of V, D, and J gene segments. The loci of these gene segments are widely separated in the germline, but the rearrangement during lymphocyte development results in the juxtaposition of V, D, and J gene segments, or V and J gene segments, and the junctions between these gene segments are characterized by small regions of nucleotide insertions and deletions (N1 and N2 regions). This process occurs randomly. or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules. Certain classes of molecular markers are very specific but present unique complexities with regard to detection, which result from gene rearrangement events. The rearrangement of genetic material in somatic cells involves bringing together two or more initially separated regions of the genome. It can occur as a random process, but it can also occur as part of the development process of normal lymphoid cells. In relation to cancer, the rearrangement can be simple or complex. A simple rearrangement may be considered one in which two unrelated genes or regions are juxtaposed. A complex rearrangement can be considered one in which three or more genes or gene segments are rearranged. A typical example of a complex rearrangement occurs during the normal development of lymphoid cells and involves the rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) variable genes with the rearrangement of V, D, and J gene segments. The loci of these gene segments are widely separated in the germline, but the rearrangement during lymphocyte development results in the juxtaposition of V, D, and J gene segments, or V and J gene segments, and the junctions between these gene segments are characterized by small regions of nucleotide insertions and deletions (N1 and N2 regions). This process occurs randomly. or are more or less heterogeneous. The level of detection of marker molecules that can be achieved depends greatly on the sensitivity and specificity of the detection method, but in most cases, as the proportion of target molecules in a larger molecular population decreases, the signal noise from the larger population makes it impossible to detect signals from the target molecules. Thus, each normal lymphocyte comes to have a unique V(D)J rearrangement that can be a complete VDJ rearrangement or a VJ or DJ rearrangement, depending on both the gene to be rearranged and the nature of the rearrangement. Lymphoid cancers such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, or myeloma arise as a result of neoplastic transformation of a single normal cell, so cancer cells will all originally have, at least, the junctional V(D)J rearrangements that were originally present in the founding cell. Subclones can arise during the expansion of the neoplastic population, and further, V(D)J rearrangements can occur among them.

[0010] The unique DNA sequences resulting from the rearrangement and present in cancer clones or subclones provide unique genetic markers that can be used to monitor the response to treatment and to determine treatment regimens. Monitoring of clones can be carried out by PCR, flow cytometry, or next-generation sequencing. Monitoring by flow cytometry involves determining the immunophenotype of cancer cells at diagnosis and, in subsequent samples, investigating the same phenotype to detect and quantify cancer cells. Next-generation sequencing is a newer approach, and its advantages and disadvantages are still being evaluated. However, this is also a costly procedure.

[0011] PCR-based analysis is a preferred method because of its potentially high level of specificity and automation. Quantification by PCR has conventionally involved sequencing of marker rearrangements using DNA from samples collected at diagnosis, synthesis of patient-specific primers, and use of these primers in PCR of DNA extracted from samples obtained during treatment. Usually, two primers ​The downstream primers are usually located on either side of the J gene segment. As an objective, an upstream primer (also known as an allele-specific oligonucleotide [ASO]) was used. (Bruggemann et al., 2004, Pongers-Willemse et al, 1999, Nakao e t al,2000, van der Velden et al,2002, van der Velden et al, 2004, van der Velden et al. al,2007, van der Velden et al,2009, van de r Velden et al, 2014, Verhagen et al, 2000) Optionally, the upstream primer targets a V gene segment and the ASO primer targets a , downstream and targeting the most variable region of the rearrangement. It is a consensus primer because it targets a conserved region common to many different rearrangements. do.

[0012] PCR-based surveillance of minimal residual disease (MRD) in leukemia is widely used in clinical practice. Usually, after the completion of induction therapy (about 1 month) and several cycles of consolidation therapy, After treatment (approximately 80 days), the number of leukemic cells (MRD) was measured to determine whether treatment should be continued or not. A decision is then made to change the MRD level at the end of induction. If the cut-off level is exceeded, a decision may be made to increase the intensity of treatment. The cut-off level varies slightly depending on the group protocol, but is usually around 10 -3 (1 / 1000)~10 -4 (1 / 10,000) of leukemia cells / total cells.

[0013] The problems that currently exist with this method are the non-specificity that gives rise to false positive results and, importantly, limits the sensitivity of detection and measurement. As a result, MRD levels below 10 cannot, in some cases, be detected and often cannot be quantified. This has two consequences. -4 · When the MRD is below the detection limit, quantification is not possible. This applies to many patients. There is great interest in attempts to identify patients who respond very well to initial treatment and whose subsequent treatment intensity can be reduced. Such patients will typically have very low levels of, for example, less than 10 MRD, but if the detection limit is 10 they cannot be distinguished from patients with levels between 10 · Due to the stochastic variation of the assay, when the MRD level is close to but still above the detection limit, the measurement accuracy is insufficient. The factors leading to non-specificity include the following. · The sequences to which the two primers bind are not unique and are usually sequences present in the genome, and amplification specificity occurs only due to rearrangements that bring the binding sequences closer together. · Some degree of homology exists between different members of the V gene family and between different members of the D gene family. This increases the likelihood that the upstream primer will hybridize to non-leukemic rearrangements. -6 MRD, but if the detection limit is 10 they cannot be distinguished from patients with levels between 10 -4 and 10 -4 to 10 -6 · Rearrangements in the population of non-leukemic lymphocytes are very heterogeneous and thus in the normal population it is not possible. · Due to the stochastic variation of the assay, when the MRD level is close to but still above the detection limit, the measurement accuracy is insufficient. The factors leading to non-specificity include the following.

[0014] · The sequences to which the two primers bind are not unique and are usually sequences present in the genome, and amplification specificity occurs only due to rearrangements that bring the binding sequences closer together. · Some degree of homology exists between different members of the V gene family and between different members of the D gene family. This increases the likelihood that the upstream primer will hybridize to non-leukemic rearrangements. · Rearrangements in the population of non-leukemic lymphocytes are very heterogeneous and thus in the normal population · The sequences to which the two primers bind are not unique and are usually sequences present in the genome, and amplification specificity occurs only due to rearrangements that bring the binding sequences closer together. · Some degree of homology exists between different members of the V gene family and between different members of the D gene family. This increases the likelihood that the upstream primer will hybridize to non-leukemic rearrangements. · Rearrangements in the population of non-leukemic lymphocytes are very heterogeneous and thus in the normal population · Rearrangements in the population of non-leukemic lymphocytes are very heterogeneous and thus in the normal population The rearrangement of one or more cells may be similar to the universal rearrangement of a population of leukemia cells .

[0015] Over the past 18 years, there have been many prior art methods that have attempted to minimize the incidence of non-specific amplification. These include . · Performing two-round or three-round nested PCR using a series of upstream primers targeting different regions of the rearranged target gene. The regions targeted usually contain two N regions. This eliminates non-specificity and results in a highly sensitive assay (e.g., Morley et al, 2009). However, this approach is more complex and there is a risk of environmental contamination by PCR products . . . . · Replacing PCR with next-generation sequencing. This procedure may be able to measure MRD at 10 -5 and perhaps, using additional steps, MRD down to 10 . However, this procedure is complex and costly, especially when high sensitivity is desired -6 . . · Using improved flow cytometry . · Designing the 3’ ends of allele-specific (ASO) primers in an attempt to minimize non-specificity. It is widely believed that it is desirable to include G or C bases at the 3’ ends of PCR primers to obtain efficient amplification. Specifically, the hybridization and extension of efficient primers is supported by the presence of one or more G or C bases at the 3’ ends. This is because these bases form stronger hydrogen bonds with complementary bases than A or T bases . . . . · Combining with an annealing temperature of about 60°C, the upstream ASO primers with the maximum N region directing a downstream primer towards the germline J sequence in the region (e.g., Bruggeman n et al, 2004, Pongers-Willemse et al, 1999 , Nakao et al, 2000, van der Velden et al, 2 002, van der Velden et al, 2004, van der Ve lden et al, 2007, van der Velden and van D ongen, 2009, van der Velden et al, 2014, Ver hagen et al, 2000). · Use of primers with a Tm of up to approximately 65°C and / or an annealing temperature of up to 69°C (Bruggemann et al, 2004, Pongers-Willem se et al, 1999, Nakao et al, 2000, van der V elden et al, 2002, van der Velden et al, 20 07, van der Velden and van Dongen, 2009, va n der Velden et al, 2014, Verhagen et al, 2 000). · Use of shortened primers. · Performing touchdown PCR (Pongers-Willemse et a l, 1999, Nakao et al 2000). · Designing primers to exhibit specific placement characteristics in relation to their placement on the rearranged gene . · Performing melting curve analysis in an effort to distinguish specific amplicons from non-specific amplicons. However, if one of the primers is a consensus primer , the melting curve analysis will fail. ​

[0016] However, these methods, which have been carried out over nearly 20 years, have not significantly reduced non-specific amplification (Bruggemann et al, 2004, Ponge rs-Willemse et al, 1999, Nakao et al, 2000, van der Velden et al, 2002, van der Velden et al, 2007, van der Velden et al, 2009, va n der Velden et al, 2014, Verhagen et al, 2 000). Indeed, non-specific amplification inevitably occurs to the extent that the criteria for interpreting MRD results recommend associating the results with the observed level of non-specificity (van der Velden et al, 2007).

[0017] Therefore, there is a continuing need to develop improved amplification methods that are simple and yet show further improved sensitivity through a reduction in the level of non-specific amplification of rearranged Ig and TCR genes, for example in the context of MRD.

[0018] In the research leading up to the present invention, highly sensitive one-round PCR has been developed based on the use of annealing temperatures determined by reactions based on the properties of primers selected for use in a given reaction. Specifically, performing reactions on the subject of annealing temperatures within the range from 3°C below the critical annealing temperature (Tc) to the Tc of the reaction results in a significant reduction in non-specific amplification that enables the quantification of MRD in samples obtained during the treatment of patients with acute lymphoblastic leukemia or chronic lymphocytic leukemia, which has not been achievable until now. is not capable. These findings are not based on annealing temperatures experimentally determined by reference to the functionality of primers selected for use in a given reaction, but rather on the selection of an annealing temperature determined as a fixed value for the entire reaction, which is in contrast to almost all prior art methods.

[0019] The design and use of primers in which one or more of the nucleotides at the 3'-end of the primer terminus are A and / or T, either alone or in combination with an annealing temperature selected according to the method of the present invention, has also been further determined to significantly reduce non-specific amplification. Also, when designed to include melt curve analysis, the method of the present invention is particularly sensitive and thus a powerful tool.

[0020] The development of the present invention is applicable to any Ig or TCR rearrangement analysis, but has been determined to show particular effectiveness with respect to the amplification of Ig or T cell rearrangement target regions containing only the N region.

[0021] These findings are unexpected and counterintuitive in light of the limited improvements achieved to date based on changes to primer design, annealing temperature, and PCR conditions, which have seen only limited success in the primers tested so far. The development of this highly sensitive method obviates the need to perform more complex multiplex or nested PCR reactions, or to use separately very expensive next-generation sequencing results. Here, the development of the method enables improved detection and / or characterization of clonal populations of lymphoid cells characterized by specific Ig or TCR gene rearrangements, such as neoplastic populations of T cells or B cells. ​​​​​​​​​​​​​​ Monitoring becomes possible. Means for diagnosing and / or monitoring a medical condition that may be characterized by the expansion of a clonal population of such cells are also provided. / Or monitoring means are also provided.

Summary of the Invention

[0022] Throughout this specification and the following claims, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", are to be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. The word "comprise", as well as variations such as "comprises" and "comprising", are to be understood to mean the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps. or group of integers or steps, but not the exclusion of any other integer or step, or group of integers or steps, will be understood to mean.

[0023] The present invention is not limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention, as described herein. Those embodiments are intended for illustrative purposes only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present invention, as described herein.

[0024] As used herein, the term "derived from" shall be construed to mean that a particular integer or group of integers is derived from a specified species, but not necessarily directly obtained from the specified source. Further, as used herein, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. but not necessarily directly obtained from the specified source. Further, as used herein, the terms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. include plural referents.

[0025] The specification of the subject matter contains nucleotide sequence information created using the PatentIn version 3.1 program presented after the references in this specification. Each nucleotide sequence In the sequence listing, a sequence is identified by a numeric heading <210> followed by a sequence identifier (e.g., <2 10>1, <210>2, etc.). The length of each nucleotide sequence, the type of sequence ( such as DNA), and the source organism are indicated by the information provided in the numeric heading columns <211>, <212>, and <213>, respectively. The nucleotide sequences referred to herein are identified by a heading "SEQ ID NO:" followed by a sequence identifier (e.g., SEQ ID NO:1, SEQ ID NO:2, etc.). The sequence identifiers referred to herein correlate with the information provided in column <400> of the sequence listing (followed by a sequence identifier (e.g., <400>1, <400>2, etc. )). That is, SEQ ID NO:1 detailed herein correlates with the sequence shown as <400>1 in the sequence listing.

[0026] One aspect of the invention relates to a method for amplifying an Ig or TCR nucleic acid region characterized by rearrangement of two or more V, D, or J gene segments, the method comprising contacting a forward and a reverse primer targeting the rearranged Ig or TCR nucleic acid region with a nucleic acid sample of interest, and amplifying the nucleic acid sample using

[0027] (i) an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or and / or (ii) at least one primer comprising at least one A and / or T nucleotide at the 3'-terminal nucleotide position, and optionally means for performing melting curve analysis.

[0027] In another aspect, a method for amplifying an Ig or TCR DNA region characterized by rearrangement of two or more V, D, or J gene segments is provided, the method comprising the rearranged Ig or TCR DNA region. Forward and reverse primers targeting the TCR nucleic acid region, contacting the nucleic acid sample of interest, and amplifying the nucleic acid sample using the following. (i) An annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or and / or (ii) At least one primer that contains at least one A and / or T nucleotide at the 3'-terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis.

[0028] In yet another aspect, a method for amplifying an Ig or TCR nucleic acid region characterized by rearrangement of two or more V, D, or J gene segments is provided, where this rearrangement is characterized by a single N region and the method comprises contacting a forward and a reverse primer targeting the rearranged Ig or TCR nucleic acid region with a nucleic acid sample of interest and amplifying the nucleic acid sample using the following. (i) An annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or and / or and / or (ii) At least one primer that contains at least one A and / or T nucleotide at the 3'-terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis.

[0029] According to these aspects, in one embodiment, the at least one primer is an ASO primer.

[0030] In another embodiment, the method is within the range between Tc and (Tc - 3°C) of the amplification reaction of interest. ​​​Annealing temperature, and one or more primers, wherein the 3' end of the nucleotide positions of the primers contains both primers containing A or T nucleotides is included.

[0031] In yet another embodiment, the nucleic acid is DNA.

[0032] In yet another embodiment, each of the two 3'-terminal nucleotide positions of the primer contains A and / or T nucleotides. In another embodiment, each of the three 3'-terminal nucleotide positions of the primer contains A and / or T nucleotides is present. In yet another embodiment, each of the four 3'-terminal nucleotide positions of the primer contains A and / or T nucleotides. In yet another embodiment, each of the five 3'-terminal nucleotide positions of the primer contains A and / or T nucleotides. In yet another embodiment, each of the six 3'-terminal nucleotide positions of the primer contains A and / or T nucleotides. is present.

[0033] In yet another embodiment, melting curve analysis is performed.

[0034] In yet another embodiment, the primer targeting the downstream gene segment targets the J segment. is targeted.

[0035] In a further embodiment, the amplification is polymerase chain reaction.

[0036] Yet another aspect of the present invention provides a method for detecting and / or monitoring a mammalian clonal cell population, wherein the clonal cells are rearrangements of two or more V, D, or J gene segments Characterized by the compilation, characterized by the Ig or TCR nucleic acid region, and the method includes the following. (i) Forward and reverse primers as described above, and DNA material of a biological sample derived from a mammal are contacted under time and conditions sufficient to facilitate the interaction between the primer and the target nucleic acid molecule. (ii) (a) Annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of the subject, and / or (b) at least one primer having at least one A and / or T nucleotide at the 3' terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis are used to amplify the nucleic acid target, and (iii) Detecting the amplification product.

[0037] According to this aspect, in one embodiment, the at least one primer is an ASO primer.

[0038] In another embodiment, the method includes an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of the subject, and both at least one primer having an A or T nucleotide at the position of the 3' terminal nucleotide of the primer.

[0039] In yet another embodiment, the nucleic acid is DNA.

[0040] In yet another embodiment, there are A and / or T nucleotides at each of the two 3' most terminal nucleotide positions of the primer. In another embodiment, there are A and / or T nucleotides at each of the three 3' most terminal nucleotide Furthermore, in another embodiment, each of the four 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the five 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the six 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide.

[0041] In yet another embodiment, melting curve analysis is performed.

[0042] In a further embodiment, the amplification reaction is a polymerase chain reaction.

[0043] In yet another embodiment, the primer targeting the downstream gene segment targets the J segment.

[0044] In a further aspect, the condition is a neoplasm, and more preferably, a lymphoid neoplasm.

[0045] In another aspect, the method of the present invention is used to detect minimal residual disease associated with lymphocytic leukemia.

[0046] Yet another aspect of the present invention relates to an isolated primer as described above.

[0047] Yet another aspect of the present invention relates to a kit for facilitating the identification of Ig or TCR nucleic acid regions characterized by the rearrangement of two or more V, D, or J gene segments, the kit comprising a portion configured to contain any one or more of the oligonucleotide primers as described above, a reagent useful for facilitating the interaction of the primer with a target nucleic acid molecule, and reagents useful for enabling the interaction to effect amplification of the nucleic acid target.

Brief Description of the Drawings

[0048]

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Mode for Carrying Out the Invention

[0049] The present invention is based, in part, on the development of a simple and sensitive amplification method for detecting a clonal cell population characterized by a rearranged Ig or TCR gene. In particular, when combined with melting curve analysis as part of the amplification process, the use of an annealing temperature within the range 3°C lower than Tc to Tc of the PCR reaction in question ("Tc - 3°C") unexpectedly enables the development of highly sensitive single-round PCR. Furthermore, the use of primers designed such that at least the terminal 3' nucleotide positions of the primers are A and / or T further improves the sensitivity and specificity of the amplification reaction, either alone or when used in combination with an annealing temperature selected according to the method of the present invention. The development of this method has facilitated the detection of specific Ig or TCR gene rearrangements of interest, particularly in the context of detecting rearrangements characterized by a unique N region. This thereby enables the detection of known Ig or TCR gene rearrangements that are expressed. detecting, and is based on the development of a simple and sensitive amplification method. In particular, when combined with melting curve analysis as part of the amplification process, the use of an annealing temperature within the range 3°C lower than Tc to Tc of the PCR reaction in question ("Tc - 3°C") unexpectedly enables the development of highly sensitive single-round PCR. Furthermore, the use of primers designed such that at least the terminal 3' nucleotide positions of the primers are A and / or T further improves the sensitivity and specificity of the amplification reaction, either alone or when used in combination with an annealing temperature selected according to the method of the present invention. The development of this method has facilitated the detection of specific Ig or TCR gene rearrangements of interest, particularly in the context of detecting rearrangements characterized by a unique N region. This thereby enables the detection of known Ig or TCR gene rearrangements that are expressed. When combined with melting curve analysis as part of the amplification process, the use of an annealing temperature within the range 3°C lower than Tc to Tc of the PCR reaction in question ("Tc - 3°C") unexpectedly enables the development of highly sensitive single-round PCR. When combined with melting curve analysis as part of the amplification process, the use of an annealing temperature within the range 3°C lower than Tc to Tc of the PCR reaction in question ("Tc - 3°C") unexpectedly enables the development of highly sensitive single-round PCR. When combined with melting curve analysis as part of the amplification process, the use of an annealing temperature within the range 3°C lower than Tc to Tc of the PCR reaction in question ("Tc - 3°C") unexpectedly enables the development of highly sensitive single-round PCR. Furthermore, the use of primers designed such that at least the terminal 3' nucleotide positions of the primers are A and / or T further improves the sensitivity and specificity of the amplification reaction, either alone or when used in combination with an annealing temperature selected according to the method of the present invention. Furthermore, the use of primers designed such that at least the terminal 3' nucleotide positions of the primers are A and / or T further improves the sensitivity and specificity of the amplification reaction, either alone or when used in combination with an annealing temperature selected according to the method of the present invention. Furthermore, the use of primers designed such that at least the terminal 3' nucleotide positions of the primers are A and / or T further improves the sensitivity and specificity of the amplification reaction, either alone or when used in combination with an annealing temperature selected according to the method of the present invention. The development of this method has facilitated the detection of specific Ig or TCR gene rearrangements of interest, particularly in the context of detecting rearrangements characterized by a unique N region. This thereby enables the detection of known Ig or TCR gene rearrangements that are expressed. It enables the improvement of the monitoring of a state characterized by the presence of cells such as a clonal population of cells. The method and primers of the present invention find a specific application in the detection of minimal residual disease that requires a high level of sensitivity and specificity, which is currently achievable only by the application of very complex and expensive molecular techniques.

[0050] Accordingly, one aspect of the present invention relates to a method for amplifying an Ig or TCR nucleic acid region characterized by the rearrangement of two or more V, D, or J gene segments, the method comprising contacting a forward and a reverse primer targeting the rearranged Ig or TCR nucleic acid region with a nucleic acid sample of interest, and amplifying the nucleic acid sample using the following. (i) An annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or (ii) At least one primer comprising at least one A and / or T nucleotide at the 3'-terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis. and / or

[0051] In one embodiment, the at least one primer is an ASO primer.

[0052] In another embodiment, the method comprises an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and using both primers, each comprising an A or T nucleotide at the 3'-terminal nucleotide position of the primer.

[0053] In yet another embodiment, melting curve analysis is performed.

[0054] References to "Ig or TCR nucleic acid regions" are to be understood as references to any region of Ig or TCR DNA or RNA for which amplification is desired. The nucleic acid region may correspond to a partially rearranged gene or a fully rearranged gene. References to "nucleic acid" or "nucleotide" or "base" are to be understood as references to both deoxyribonucleic acid or nucleotides and ribonucleic acid or nucleotides or purine or pyrimidine bases or derivatives or analogs thereof. In this regard, it is to be understood to include, inter alia, DNA (cDNA or genomic DNA), RNA or mRNA, and phosphate esters of ribonucleotides and / or deoxyribonucleotides. The nucleic acid molecules of the present invention can be of any origin, including naturally occurring ones (e.g., those that would be derived from a biological sample), recombinantly produced ones, or synthetically produced ones. The base can also be a non-standard base such as inosine. References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall

[0055] References to "Ig or TCR nucleic acid regions" are to be understood as references to any region of Ig or TCR DNA or RNA for which amplification is desired. The nucleic acid region may correspond to a partially rearranged gene or a fully rearranged gene. References to "nucleic acid" or "nucleotide" or "base" are to be understood as references to both deoxyribonucleic acid or nucleotides and ribonucleic acid or nucleotides or purine or pyrimidine bases or derivatives or analogs thereof. In this regard, it is to be understood to include, inter alia, DNA (cDNA or genomic DNA), RNA or mRNA, and phosphate esters of ribonucleotides and / or deoxyribonucleotides. The nucleic acid molecules of the present invention can be of any origin, including naturally occurring ones (e.g., those that would be derived from a biological sample), recombinantly produced ones, or synthetically produced ones. The base can also be a non-standard base such as inosine. References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "Ig or TCR nucleic acid regions" are to be understood as references to any region of Ig or TCR DNA or RNA for which amplification is desired. The nucleic acid region may correspond to a partially rearranged gene or a fully rearranged gene. References to "nucleic acid" or "nucleotide" or "base" are to be understood as references to both deoxyribonucleic acid or nucleotides and ribonucleic acid or nucleotides or purine or pyrimidine bases or derivatives or analogs thereof. In this regard, it is to be understood to include, inter alia, DNA (cDNA or genomic DNA), RNA or mRNA, and phosphate esters of ribonucleotides and / or deoxyribonucleotides. The nucleic acid molecules of the present invention can be of any origin, including naturally occurring ones (e.g., those that would be derived from a biological sample), recombinantly produced ones, or synthetically produced ones. The base can also be a non-standard base such as inosine. References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "Ig or TCR nucleic acid regions" are to be understood as references to any region of Ig or TCR DNA or RNA for which amplification is desired. The nucleic acid region may correspond to a partially rearranged gene or a fully rearranged gene. References to "nucleic acid" or "nucleotide" or "base" are to be understood as references to both deoxyribonucleic acid or nucleotides and ribonucleic acid or nucleotides or purine or pyrimidine bases or derivatives or analogs thereof. In this regard, it is to be understood to include, inter alia, DNA (cDNA or genomic DNA), RNA or mRNA, and phosphate esters of ribonucleotides and / or deoxyribonucleotides. The nucleic acid molecules of the present invention can be of any origin, including naturally occurring ones (e.g., those that would be derived from a biological sample), recombinantly produced ones, or synthetically produced ones. The base can also be a non-standard base such as inosine. References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall

[0056] References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall References to "derivative" are to be understood to include references to fragments, portions, parts, homologs, and mimetics of the nucleic acid molecule from natural, synthetic, or recombinant sources. "Functional derivative" is to be understood as a derivative that exhibits one or more functional activities of a purine or pyrimidine base, nucleotide, or nucleic acid molecule. Derivatives of the nucleotide or nucleic acid sequence include fragments having a particular region of the nucleotide or nucleic acid molecule fused to other proteinaceous or non-proteinaceous molecules. "Analog" as contemplated herein refers to modifications to the nucleotide or nucleic acid molecule, e.g., to its chemical composition or overall including, but not limited to, modifications to the three-dimensional configuration. These include, for example, modifications to the way in which nucleotides or nucleic acid molecules interact with other nucleotides or nucleic acid molecules, such as at the level of backbone formation or complementary base pair hybridization. Locked nucleic acids are examples of the analogs described herein. Biotinylation of nucleotides or nucleic acid molecules is an example of the "functional derivatives" described herein. Derivatives of nucleic acid molecules can result from substitution, deletion, and / or addition of single or multiple nucleotides. The term "functional derivative" is also understood to encompass any one or more of the functional activities of a nucleotide or nucleic acid sequence, such as a product obtained after screening natural products, that is exhibited by a nucleotide or nucleic acid.

[0057] The "nucleic acid" region of the subject matter can be DNA or RNA or derivatives or analogs thereof. Since the region of interest is a DNA sequence encoding a proteinaceous molecule, it can take the form of genomic DNA, cDNA generated from an mRNA transcript, or DNA generated by nucleic acid amplification. Where the method of the subject matter relates to detecting a region of RNA, it will be understood that it is necessary to first reverse transcribe the RNA to DNA, for example, using RT-PCR. The RNA of the subject matter can be any form of RNA, such as mRNA, primary RNA transcript, ribosomal RNA, transfer RNA, microRNA, etc. Preferably, the nucleic acid region of interest is a DNA region of interest. For this purpose, the DNA includes DNA generated by reverse transcription from the RNA that is ultimately the subject of the analysis, and DNA generated by any nucleic acid amplification method such as PCR. ​

[0058] The nucleic acid region to be amplified is two or more of the V, D, or J gene segments that have undergone rearrangement in an Ig or TCR nucleic acid region. Thus, the nucleic acid region of the subject of interest can correspond to either a partially or fully rearranged gene. As will be discussed in more detail below, Ig and TCR rearrangements occur as a series of successive rearrangements, resulting in, at the final step, a fully rearranged variable region that is rearranged to bind to the constant region gene. The method of the present invention may amplify all or part of the partially rearranged gene depending on the point at which the clonal lymphoid cells can arrest differentiation. In one embodiment, the target nucleic acid region is DNA. According to this embodiment, there is provided a method for amplifying an Ig or TCR DNA region characterized by rearrangement of two or more V, D, or J gene segments, the method comprising contacting a forward and reverse primer targeting the rearranged Ig or TCR nucleic acid region with a nucleic acid sample of interest, and amplifying the nucleic acid sample using:

[0059]

[0060] (i) an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of the subject, and / or (ii) at least one primer comprising at least one A and / or T nucleotide at the 3'-terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis.

[0061] In another embodiment, the at least one primer is an ASO primer.

[0062] In yet another embodiment, the method is within the range between Tc and (Tc - 3°C) of the amplification reaction of the subject, and at the position of the 3'-terminal nucleotide of the primer n, using both at least one primer containing an A or T nucleotide. .

[0063] In yet another embodiment, melting curve analysis is performed.

[0064] The method of the present invention is achieved by contacting a forward and a reverse primer with a nucleic acid sample to be tested. The reference to "primer" or "oligonucleotide primer" should be understood as a reference to any molecule including a nucleotide sequence, or a functional derivative or analog thereof, the function of which includes hybridization to a region of the nucleic acid molecule of interest. The primer may contain one or more locked nucleic acids. It should also be understood that the primer may contain non-nucleic acid components. For example, the primer may also contain non-nucleic acid tags such as fluorescent or enzyme tags, or other non-nucleic acid components that facilitate the use of the molecule as a probe or otherwise facilitate separate detection or immobilization. The primer may also contain additional nucleic acid components such as oligonucleotide tags. In another example, the primer may be a peptide nucleic acid containing a peptide backbone representing the side chain of the nucleic acid. Preferably, the oligonucleotide primer is a DNA primer. The primer of the present invention "targets" the rearranged Ig or TCR nucleic acid. "Targets" means that the primer is the rearranged Ig or TCR nucleic acid required to be amplified. means being designed to hybridize partially or wholly to the region.

[0065] Without limiting the present invention to any one theory or mode of action, V(D)J recombination in organisms having an adaptive immune system is an example of a type of site-specific gene rearrangement that helps immune cells rapidly diversify to recognize and adapt to new pathogens. Each lymphocyte undergoes somatic rearrangement of germline variable region gene segments (either V and J, D and J, or V, D, and J segments) according to specific rearranged gene segments that give rise to a total antigen diversity of approximately 10 different variable region structures. In any given lymphocyte, such as a T cell or a B cell, at least two different variable region gene segment rearrangements can occur due to rearrangements of two or more of the chains that make up the TCR or immunoglobulin molecule, specifically, the α, β, γ, or δ chains of the TCR and / or the heavy and light chains of the immunoglobulin molecule. In addition to the rearrangement of the VJ, DJ, or VDJ segments of any given immunoglobulin or TCR gene, nucleotides are randomly removed and / or inserted at the junctions between segments. This leads to the generation of an enormous diversity. Although the loci of these gene segments are widely separated in the germline, during lymphocyte development, rearrangement brings the V, (D), and J genes into juxtaposition, and the junctions between these genes are characterized by small regions of nucleotide insertion and deletion. This process occurs randomly so that each normal lymphocyte has a unique V(D)J rearrangement. Acute 16

[0066] Lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, or myeloma, such as lymphatic cancer, results from the neoplastic transformation of a single normal cell, so all cancer cells will originally have at least the junctional V(D)J rearrangement that originally exists in the founder cells. Subclones can occur during the growth of the neoplastic population, and furthermore, V(D)J rearrangement can occur among them.

[0067] References to "gene segments" should be understood as references to the V, D, and J regions of immunoglobulin and T cell receptor genes. The V, D, and J gene segments are clustered into families. For example, there are 52 different functional V gene segments and 5 J gene segments of the κ immunoglobulin light chain. In the immunoglobulin heavy chain, there are 5 5 functional V gene segments, 23 functional D gene segments, and 6 J gene segments. There are numerous individual gene segments throughout the V, D, and J gene segment families of immunoglobulins as well as T cell receptors, thereby allowing for an enormous diversity of unique combinations of V(D)J rearrangements that can be affected. For clarity, the rearranged immunoglobulin or T cell receptor [V(D)J variable nucleic acid region is referred to herein as a "gene", and the individual V, D, or J nucleic acid regions are referred to as "gene segments". Thus, the term "gene segment" is not a reference to segments of a gene per se. Rather, in the context of Ig and TCR gene rearrangements, it is a reference to a gene in connection with the clustering of these gene segments into families. The "rearranged" immunoglobulin or T cell receptor variable region gene In the context, it is a reference to a gene in connection with the clustering of these gene segments into families. A gene, as used herein, is understood to be two or more of one V segment, one J segment, and one D segment (when the D segment is incorporated into a particular rearrangeable variable gene in question) spliced together to form a single rearranged "gene". In reality, this rearranged "gene" is actually a stretch of genomic DNA containing one V gene segment, one J gene segment, and one D gene segment that are spliced together. Thus, in reality, it is actually composed of two or three different V, D, or J genes (referred to as gene segments herein) that are spliced together, and may be referred to as a "gene region" (although not in the context of this specification). Therefore, the individual "gene segments" of the rearranged immunoglobulin or T cell receptor genes are described as individual V, D, and J genes. These genes are described in detail in the IMGT database. The term "gene" will be used herein to refer to a rearranged immunoglobulin or T cell receptor variable gene. The term "gene segment" will be used herein to refer to V, D, J, and framework 3 regions. However, it should be noted that there is considerable inconsistency in the use of the terms "gene" / "gene segment" with respect to immunoglobulin and T cell receptor rearrangement. For example, IMGT refers to individual V, D, and J "genes", while some scientific publications refer to these as "gene segments". There are also suppliers who refer to the rearranged variable immunoglobulin or T cell receptor as a "gene region", as well as those who refer to it as a "gene". The terms used herein are as follows. (when incorporated into) two or more of one V segment, one J segment, and one D segment are spliced together to form a single rearranged "gene". It should be understood as a gene. In fact, this rearranged "gene" is actually a stretch of genomic DNA containing one V gene segment, one J gene segment, and one D gene segment that are spliced together. Therefore, in fact, two or three different V, D, or J genes (referred to as gene segments herein) that are spliced together actually constitute it, and may be referred to as a "gene region" (although not in the context of this specification). Therefore, the individual "gene segments" of the rearranged immunoglobulin or T cell receptor gene are described as individual V, D, and J genes. These genes are described in detail in the IMGT database. The term "gene" will be used herein to refer to a rearranged immunoglobulin or T cell receptor variable gene. The term "gene segment" will be used herein to refer to V, D, J, and framework 3 regions. However, with respect to immunoglobulin and T cell receptor rearrangement, it should be noted that there is considerable inconsistency in the use of the terms "gene" / "gene segment". For example, IMGT refers to individual V, D, and J "genes", while some scientific publications refer to these as "gene segments". There are also suppliers who refer to the rearranged variable immunoglobulin or T cell receptor as a "gene region", as well as those who refer to it as a "gene". The terms used herein are as described above. "genes", while some scientific publications refer to these as "gene segments". There are also suppliers who refer to the rearranged variable immunoglobulin or T cell receptor as a "gene region", as well as those who refer to it as a "gene". The terms used herein are as follows. is as follows.

[0068] Furthermore, without limiting the present invention to any one theory or mode of action, gene re- The nature of the recombination event is characterized by deletions and insertions of random nucleotides at the junction between the recombined gene or gene segment (described herein) that result in the formation of an "N region". such that the junction results in deletions and insertions of random nucleotides that result in the formation of an "N region". These N regions are also unique and thus are useful targets in connection with the design of the primers of the present invention. In this regard, with respect to the sub-regions of the primers of the present invention, for purposes of simplifying the discussion herein as compared to the gene / gene segment to which they bind, these N regions, although they do not exist on the chromosome as separate gene segments and are only generated during rearrangement by nucleotide insertions and deletions at the recombination site, may alternatively be referred to in the context of the present invention as "gene segments". The choice of referring to these as N gene segments or N regions is made purely for purposes of simplifying the language in any given section of this document for purposes of clarity. Thus, in particular, in the context of V(D)J recombination, the gene segments that can be the subject of analysis are the individual V, N1, D, N2, and J regions, as well as the framework 3 gene segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. segments. In this context, the N gene segment between the V and D gene segments is referred to as N1, and the N gene segment between the D and J gene segments is referred to as N2. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. However, it should also be understood that an N region (or "gene segment") can occur within a V, D, or J gene segment. Without limiting the present invention to any one theory of mode of action, this can occur within the D gene segment during recombination with the J gene segment. is most commonly observed in relation to D gene segments that can undergo the formation of one or more N regions Thus, a fully rearranged VDJ region will generally always contain N1 and N2 gene segments, although it may also contain additional N regions, for example, within a V, D, or J gene segment, or even at the junction of the rearranged J gene segment and the constant gene, and this final rearrangement usually occurs after the completion of VDJ rearrangement.

[0069] A reference to a "forward primer" (or "upstream primer") is to be understood as a primer that hybridizes to the antisense strand of the target DNA and to the 5' of other primers to amplify the target nucleic acid (e.g., DNA) in a nucleic acid (e.g., DNA) sample of interest.

[0070] A reference to a "reverse primer" (or "downstream primer") is to be understood as a primer that hybridizes to the sense strand of the target nucleic acid (e.g., DNA) or to the 3' of other primers to amplify the target nucleic acid (e.g., DNA) in a nucleic acid (e.g., DNA) sample of interest and in PCR.

[0071] Generally, the forward or upstream primer is an ASO primer, and the downstream or reverse primer is, for example, a consensus primer targeting the conserved J region, although in some situations, conversely, for example, the forward primer targets a conserved sequence in the V region, and the reverse primer is an ASO primer.

[0072] Means for designing and synthesizing primers suitable for use in the present invention are known to those skilled in the art​​ will be well known. As detailed above, the V, D, and J gene segment families have been fully identified and sequenced. Thus, the design of primers that amplify specific segments or combinations of rearranged segments is well within the skill of those in the art. Nevertheless, substantial exemplification is also provided in Examples 1 and 2 in connection with the design and testing of primers that exhibit the Tc values referred to herein. As detailed above, the method of the present invention is based on the determination that a significantly improved level of sensitivity is achieved when the annealing temperature of the amplification reaction is selected by reference to the critical annealing temperature (which is specific to each reaction as defined by the particular primer set). Reference to the "critical annealing temperature" ("Tc") is to be understood as reference to the highest annealing temperature at which an amplification reaction, such as PCR, operates with optimal efficiency with respect to the amplification of the desired target. The efficiency of the amplification operation can be evaluated by Ct. Specifically, optimal amplification in each cycle of PCR depends on the optimal hybridization of each primer to the target molecule such that substantially all target molecules become hybridized. Without limiting the present invention to any theory or mode of action, when a series of amplification reactions are carried out using a variety of different but gradually increasing annealing temperatures, a temperature will be identified at which hybridization becomes incomplete, amplification becomes inefficient, and Ct increases. The highest temperature before these amplification inefficiencies become apparent is the critical annealing temperature (Tc), as shown in Figure 3. The Tc of a primer is an empirical value that is several degrees different from the calculated Tm.

[0073] As detailed above, the method of the present invention is based on the determination that a significantly improved level of sensitivity is achieved when the annealing temperature of the amplification reaction is selected by reference to the critical annealing temperature (which is specific to each reaction as defined by the particular primer set). Reference to the "critical annealing temperature" ("Tc") is to be understood as reference to the highest annealing temperature at which an amplification reaction, such as PCR, operates with optimal efficiency with respect to the amplification of the desired target. The efficiency of the amplification operation can be evaluated by Ct. Specifically, optimal amplification in each cycle of PCR depends on the optimal hybridization of each primer to the target molecule such that substantially all target molecules become hybridized. Without limiting the present invention to any theory or mode of action, when a series of amplification reactions are carried out using a variety of different but gradually increasing annealing temperatures, a temperature will be identified at which hybridization becomes incomplete, amplification becomes inefficient, and Ct increases. The highest temperature before these amplification inefficiencies become apparent is the critical annealing temperature (Tc), as shown in Figure 3. The Tc of a primer is an empirical value that is several degrees different from the calculated Tm. Specifically, optimal amplification in each cycle of PCR depends on the optimal hybridization of each primer to the target molecule such that substantially all target molecules become hybridized. Without limiting the present invention to any theory or mode of action, when a series of amplification reactions are carried out using a variety of different but gradually increasing annealing temperatures, a temperature will be identified at which hybridization becomes incomplete, amplification becomes inefficient, and Ct increases. The highest temperature before these amplification inefficiencies become apparent is the critical annealing temperature (Tc), as shown in Figure 3. The Tc of a primer is an empirical value that is several degrees different from the calculated Tm. When a series of amplification reactions are carried out using a variety of different but gradually increasing annealing temperatures, a temperature will be identified at which hybridization becomes incomplete, amplification becomes inefficient, and Ct increases. The highest temperature before these amplification inefficiencies become apparent is the critical annealing temperature (Tc), as shown in Figure 3. The Tc of a primer is an empirical value that is several degrees different from the calculated Tm. ​It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets.

[0074] Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets. It will be well understood by those skilled in the art that it is the temperature determined in a certain way. The magnitude of this difference will vary for each primer. Furthermore, since the exact value of Tc depends on the resolution of the temperature gradient generated by the equipment used to determine the amplification conditions and Tc, it is understood by those skilled in the art that the amplification conditions under which Tc is calculated must correspond to the amplification conditions subsequently used for amplifying the test sample. The determination of the Tc value is well within the skill of those skilled in the art and can be carried out by any suitable means, for example, using an instrument that enables real-time amplification of the target DNA over a range of annealing temperatures. An example of the determination of Tc is described in Example 2. Since the determination of Tc is relevant to the analysis of the actual performance of the primers whose usefulness has been selected, it should also be understood that it needs to be evaluated in the context of the specific primers proposed to be used for amplifying the test sample. Therefore, this analysis will need to be completed before the method of the present invention is applied and will need to be carried out in the context of each different set of primers proposed to be used. However, as detailed above, the method for determining Tc is well-known and can be carried out routinely and easily without any undue burden. For this purpose, the reference to the annealing temperature selected for the "subject reaction" of Tc should be understood as a reference to the fact that the relevant Tc is the Tc of the specific primers required to be used in the context of a given reaction. Therefore, Tc is not a fixed value that can be applied to various different primer sets.

[0075] References to "annealing temperature" should be understood as referring to the temperature of the amplification step at which primer hybridization to the target nucleic acid region (template) occurs. As detailed above, one aspect of the invention is based on the determination that reduction of non-specific amplification can be achieved when the annealing temperature used falls from Tc to 3°C below Tc (referred to herein as "Tc - 3°C"). Therefore, the annealing temperature of the method of the invention may vary from reaction to reaction if different primers are used. This design is completely different from most amplification systems where a fixed annealing temperature is used across all reactions. Further, references to annealing temperatures that "fall within" or "between" Tc and (Tc - 3°C) should be understood to include the option of using the Tc or (Tc - 3°C) temperature itself. It should also be understood that the use of temperatures outside or near these limits, for example, temperatures that are slightly below or above these limits but function equivalently to temperatures within this range, is also contemplated. As detailed above, one aspect of the invention is based on the determination that reduction of non-specific amplification can be achieved when the annealing temperature used falls from Tc to 3°C below Tc (referred to herein as "Tc - 3°C"). As detailed above, one aspect of the invention is based on the determination that reduction of non-specific amplification can be achieved when the annealing temperature used falls from Tc to 3°C below Tc (referred to herein as "Tc - 3°C"). As detailed above, one aspect of the invention is based on the determination that reduction of non-specific amplification can be achieved when the annealing temperature used falls from Tc to 3°C below Tc (referred to herein as "Tc - 3°C"). Therefore, the annealing temperature of the method of the invention may vary from reaction to reaction if different primers are used. This design is completely different from most amplification systems where a fixed annealing temperature is used across all reactions. Therefore, the annealing temperature of the method of the invention may vary from reaction to reaction if different primers are used. This design is completely different from most amplification systems where a fixed annealing temperature is used across all reactions. Therefore, the annealing temperature of the method of the invention may vary from reaction to reaction if different primers are used. This design is completely different from most amplification systems where a fixed annealing temperature is used across all reactions. Therefore, the annealing temperature of the method of the invention may vary from reaction to reaction if different primers are used. This design is completely different from most amplification systems where a fixed annealing temperature is used across all reactions. Further, references to annealing temperatures that "fall within" or "between" Tc and (Tc - 3°C) should be understood to include the option of using the Tc or (Tc - 3°C) temperature itself. Further, references to annealing temperatures that "fall within" or "between" Tc and (Tc - 3°C) should be understood to include the option of using the Tc or (Tc - 3°C) temperature itself. It should also be understood that the use of temperatures outside or near these limits, for example, temperatures that are slightly below or above these limits but function equivalently to temperatures within this range, is also contemplated. It should also be understood that the use of temperatures outside or near these limits, for example, temperatures that are slightly below or above these limits but function equivalently to temperatures within this range, is also contemplated.

[0076] Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. The ASO primer is the most important primer in terms of minimizing non-specificity, and in fact, the same reverse consensus (usually J) primer will often be used in conjunction with various ASO primers when testing samples from various patients. In this situation, it is possible that the reverse primer can be designed first to obtain a specific Tm, and then the Tc can be determined experimentally. Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. Furthermore, since the forward and reverse primers have different sequences, it will be understood that the Tc of the two primers within a single reaction may vary somewhat. Those skilled in the art will understand that this can be done. Thereafter, an ASO primer is designed to obtain a Tm that is 1-2 °C lower than that of the reverse primer. Next, the Tc of the ASO primer must be slightly lower than the Tc of the reverse primer, and the annealing temperature of PCR will optimize the specificity of PCR if it is based on the Tc of the ASO primer.

[0077] As detailed above, the forward primer is usually an ASO primer and targets the most variable regions of IgH or TCR rearrangement, and the reverse primer targets the downstream gene segments of the IgH or TCR gene. Since the improvement in sensitivity obtained by the present invention is due to the reduction of non-specificity, it is also advantageous to design the above-mentioned non-ASO primers to further minimize non-specificity.

[0078] As described above, in a further aspect, those skilled in the art may attempt to design and synthesize primers to enable optimal hybridization, for example, by inserting one or more A and / or T nucleotides at the 3' end of the primer. In one embodiment where two or more A and / or T nucleotides are utilized, they are arranged directly adjacent to each other. That is, they are arranged in sequence. Specifically, preferably, at least one A or T nucleotide may be included at the 3' terminal nucleotide position of one or both of the forward or reverse primers. In another example, the A or T nucleotide is ​​​​​​​​​​​​​ In this case, at each of the two 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the three 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the four 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the five 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the six 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. The reference to the "3'-end" of the primer is to be understood as a reference to the end of the primer that will undergo extension when hybridizing to the target. Therefore, the 1, 2, 3, 4, 5, or 6 nucleotides referred to in this context are the terminal nucleotides of the primer at the 3'-end, before the primer undergoes extension. Any combination of A or T nucleotides may be used at any one or more of these positions.

[0079] In one embodiment, at the 3'-terminal nucleotide position of the primer, A or T nucleotides are present. In another embodiment, at each of the two 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the three 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the four 3'-terminal In each case, A and / or T nucleotides are present. In yet another embodiment, at each of the five 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present. In yet another embodiment, at each of the six 3'-terminal nucleotide positions of the primer, A and / or T nucleotides are present.

[0080] As detailed above, when the amplification is designed to incorporate melt curve analysis, yet another sensitivity can be achieved. Reference to "melt curve analysis" should be understood as a reference to the analysis of the melting point of the amplicon as a means of identifying non-specific amplification. Since the melting point of the amplicon is related to the composition and length of the nucleotide sequence of the amplicon, melt curve analysis can be used to detect length and / or sequence variations within a sample, particularly to distinguish the generation of specific amplicons from the generation of non-specific amplicons. Means for performing melt curve analysis after completion of PCR are well known to those skilled in the art and are routinely incorporated into amplification reactions. Any suitable method can be utilized, such as emitting fluorescence when binding non-specifically to DNA duplexes or incorporation into a separately detectable reaction molecule. Without limiting the present invention to any one theory or mode of action, when melt curve analysis is performed by gradually increasing the temperature, the fluorescence decreases gradually over a wide range of temperatures due to the heterogeneous melting of the It will be recognized. Peaks will be observed at specific temperatures. Nonspecific amplification usually does not produce peaks, or produces complex patterns, or produces peaks at temperatures different from those of the target amplicon. or produces peaks at temperatures different from those of the target amplicon.

[0081] Furthermore, the incorporation of the probe coupled to the detection means into the method of the present invention is such that the sequence of the probe is complementary to the sequence of the target amplicon, thereby binding almost completely to the intended target, providing additional specificity. Molecules that fluoresce when binding nonspecifically to DNA duplexes are used in endpoint Ct measurements in PCR, but since they bind to all DNA duplexes, they are contraindicated for this purpose in the present method, and their Ct values are not reliable when attempting to measure low levels of MRD. Therefore, it is recommended that such fluorophores be used only for melting curve analysis. However, such fluorophore molecules can be used in the method for melting point analysis of DNA duplexes present after completion of PCR, provided that the nature and concentration of the specific fluorophore used do not affect the efficiency of PCR amplification as indicated by the lack of effect on the Ct values provided by the probe. An example of melting curve analysis using the fluorophore Syto82 to distinguish specific amplification from nonspecific amplification is shown in FIG. 4. thereby binding almost completely to the intended target, providing additional specificity. Molecules that fluoresce when binding nonspecifically to DNA duplexes are used in endpoint Ct measurements in PCR, but since they bind to all DNA duplexes, they are contraindicated for this purpose in the present method, and their Ct values are not reliable when attempting to measure low levels of MRD. Therefore, it is recommended that such fluorophores be used only for melting curve analysis. However, such fluorophore molecules can be used in the method for melting point analysis of DNA duplexes present after completion of PCR, provided that the nature and concentration of the specific fluorophore used do not affect the efficiency of PCR amplification as indicated by the lack of effect on the Ct values provided by the probe. An example of melting curve analysis using the fluorophore Syto82 to distinguish specific amplification from nonspecific amplification is shown in FIG. 4. as indicated by the lack of effect on the Ct values provided by the probe. An example of melting curve analysis using the fluorophore Syto82 to distinguish specific amplification from nonspecific amplification is shown in FIG. 4.

[0082] Therefore, the probe provides Ct and a dye, or other suitable means enables melting analysis. It will be understood by those skilled in the art that the probe can be designed to bind to any suitable detection means. However, in the context of the methods exemplified herein, or other suitable means enables melting analysis. It will be understood by those skilled in the art that the probe can be designed to bind to any suitable detection means. However, in the context of the methods exemplified herein, ​​Both the probe and the dye fluoresce in response to UV, but since their emission spectra are different , they can be monitored separately.

[0083] Thus, in yet another embodiment, the method preferably includes a Ct determination using a probe directed to the rearranged Ig or T CR of interest, which is bound to the detection means .

[0084] The method of the present invention is particularly useful in connection with improving the sensitivity of detecting rearrangement of Ig or TCR genes containing only a single N region (or also referred to herein as a "gene segment") . Without limiting the invention to any one theory or mode of action , both complete Ig heavy chain gene rearrangement or complete TCRβ chain gene rearrangement will include VDJ rearrangement , but will usually contain two N regions (N1 and N2). However, partial rearrangements may contain one or two N regions, depending on whether one or more of the V, D, or J gene segments exhibit internal N regions formed during the rearrangement process. For example, Ig light chains and TCRα chains contain only VJ rearrangement, but partial DJ rearrangement of IgH chains and TCRβ chains occurs before the rearrangement of the V gene segment by DJ rearrangement is complete. Thus, V DJ rearrangement generates two N regions (or three or more if N regions within the V, D, or J gene segments are generated), which are configured as VN1DN2J, and Ig light chains, TCRα chains, or transient partial rearrangements may generate only a single N region such as VNJ or DNJ . This can apply to loci such as IGκ and TCRγ that do not contain a D gene, and rearrangement includes only the V gene and the J gene . This is VJ or also can generate a single N region . This can apply to loci such as IGκ and TCRγ that do not contain a D gene, and rearrangement includes only the V gene and the J gene. This is VJ or IGH may have only "partial" rearrangements that have a DJ structure and a unique N region or may also apply to rearrangements of the TCRβ gene.

[0085] Thus, in another aspect, provided is a method for amplifying an Ig or TCR nucleic acid region characterized by rearrangement of two or more V, D, or J gene segments, wherein the rearrangement is characterized by a unique N region, and the method comprises contacting a forward and a reverse primer to the rearranged Ig or TCR nucleic acid region and the nucleic acid sample of interest, and amplifying the nucleic acid sample using (i) an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or (ii) at least one primer that contains at least one A and / or T nucleotide at the 3' terminal nucleotide position, and optionally, means for performing melting curve analysis. (i) an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and and / or (ii) at least one primer that contains at least one A and / or T nucleotide at the 3' terminal nucleotide position, and optionally, means for performing melting curve analysis.

[0086] In one embodiment, the at least one primer is an ASO primer.

[0087] In another embodiment, the method comprises using both an annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest and at least one primer that contains an A or T nucleotide at the 3' terminal nucleotide position of the primer. In yet another embodiment, the nucleic acid is DNA. In yet another embodiment, each of the two 3' most terminal nucleotide positions of the primer

[0088] In yet another embodiment, the nucleic acid is DNA.

[0089] In yet another embodiment, each of the two 3' most terminal nucleotide positions of the primer In addition, A and / or T nucleotides are present. In yet another embodiment, at each of the three 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present. In yet another embodiment, at each of the four 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present. In yet another embodiment, at each of the five 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present. In yet another embodiment, at each of the six 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present. In yet another embodiment, at each of the six 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present. In yet another embodiment, at each of the six 3'-terminal nucleotide positions of the primer A and / or T nucleotides are present.

[0090] In yet another embodiment, melting curve analysis is performed.

[0091] In yet another embodiment, the method preferably includes a Ct determination using a probe targeting the rearranged Ig or TCR of interest, and this probe is bound to the detection means. In yet another embodiment, the primer targeting the downstream gene segment targets the J segment.

[0092] Examples of J primers suitable for use in this embodiment of the invention are provided in Table 1 below as follows.

[0093] Table 1: J primers used for IGH and TCRβ. These are the longest primers used in these regions. Shorter primers are also used, including the same intermediate and 3'-ends of the above primers, but the shorter primers have variable 5'-bases removed to yield primers with the desired Tm. as follows.

Table 1

[0094] According to the embodiments of the present invention described herein, primers that hybridize to one or more N gene segments, such as the N1 gene segment at the 5' of the D gene segment of the fully rearranged VDJ gene, the N2 gene segment at the 3' of the D gene segment, or the N region (gene segment) that can be formed within the rearranged V, D, or J gene segment, may be designed. In this regard, in one embodiment, a high level of sensitivity is achieved when at least one of the forward or reverse primers hybridizes to at least two N gene segments. In a preferred embodiment, the forward (ASO) primer hybridizes to two N gene segments. In this embodiment, the reverse primer may hybridize to a downstream gene segment such as the J gene segment and does not necessarily have to be patient-specific. In another embodiment, the reverse primer hybridizes to at least two N gene segments. It should also be recognized that there may be situations where it is preferable for the primer to target only a single N region, even though the VDJ rearrangement contains two or more N regions. Such situations can occur when the targeted N region is particularly long, enabling high specificity, or when the targeted N region contains a sufficient number of A or T bases to allow the 3' end of the primer to contain complementary T or A bases, or when the non-targeted N regions contain an insufficient number of A or T bases. Attempts are made to design primers that hybridize between two N gene segments.

[0095]

[0096] ​​​​​​​​​​​​​​​ Within this range, the primer design of the present invention must hybridize between two N gene segments located at the junction of both V, D, or J gene segments such that the primer must hybridize between at least three consecutive gene segments of the target rearranged gene, and the 5' and 3' gene segments of these three consecutive gene segments are N regions / gene segments. Thus, the primer is configured to include three sub-regions each designed to hybridize to one of the three consecutive gene segments of the target rearranged gene, two of which are N gene segments. By "consecutive" is meant that the three gene segments are preferably rearranged such that they are directly adjacent to each other in a linear arrangement. In this regard, it should be understood that each of the three sub-regions of the primer that hybridize to the three rearranged and consecutive gene segments are operably linked to each other to form a single primer. The primer sub-regions may be the same or different in length. Depending on the length of these oligonucleotide sub-regions and the specific gene segment targets they are directed to, it should be understood that they may hybridize exclusively to all or part of just one gene segment, or they may hybridize to all or part of each of several gene segments, depending on their respective lengths. Conceptually, primers for partial rearrangement (DNJ or VNJ) can be designed to hybridize to, for example, one of the following rearranged gene segment targets: N,

[0097] gene segments: N, ​ DN, NJ, DNJ, VN, NJ, VNJ. Primers for complete VN1DN2J rearrangement - can be designed to hybridize to, for example, one of the following rearranged gene segment targets: N1, VN1, N1D, VN1D, N1DN2, VN1DN2, N1 DN2J, VN1DN2J, N2, DN2, N2J, DN2J. It should be understood that the V, D, and J gene segments may also contain an N region within the segment itself. As will be appreciated, when a primer is designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding

[0098] As will be understood, when a primer is designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding - can be designed to hybridize to two N gene segments, the primer will span the entire length of the gene segment intervening between the two N gene segments. The normal situation is VDJ rearrangement, and the intervening gene segment is usually the D gene segment. Due to the length of the D gene segment, designing a primer that hybridizes across the entire length of the D gene segment can result in loss of specificity because the sub-region of the primer that hybridizes to the D gene segment is likely to represent the longest sub-region of the primer, as will be understood by those skilled in the art. Therefore, most of the hybridization of the primer is determined by complementarity to the D gene segment, and the specificity of the N gene segment can be compromised. The particular D gene segment of interest may be present in many other VDJ rearrangement combinations in addition to the unrearranged gene, and therefore it may be detected in many different cells other than the cell of interest. This can lead to significant false positive results. The incidence of this non-specific binding To reduce it (regardless of the number of N regions targeted by the primer), the specificity can be reduced by making changes to the sub-region of the primer that hybridizes to the D gene segment. The methods to achieve this include, but are not limited to, the following. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. The methods to achieve this include, but are not limited to, the following. (i) Introduction of one or more spacers or linkers to reduce the hybridization of the oligonucleotide sub-region of the primer designed to hybridize to the D gene segment (or other gene segment for which specificity reduction is attempted) to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. (ii) Synthesis of a primer (referred to as an N mixture) in which the addition of a single nucleotide at several nucleotide positions within the sub-region of the primer designed to hybridize to a gene segment such as the D gene segment is replaced by the addition of a nucleotide randomly selected from an equimolar mixture of all four nucleobases. This significantly reduces the contribution of hydrogen bonds at these positions to the overall hybridization of the oligonucleotide primer because the probability of proper binding between the template nucleotide and the oligonucleotide nucleotide is only 1 / 4 at each round of amplification at the substitution position. Using an N mixture with low randomness, for example, using only A / T, results in a lower effect. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. To reduce it, changes can be made to the sub-region of the primer that hybridizes to the D gene segment. .

[0099] Without limiting the present invention to any theory or mode of action, it has been determined that modifying with an N mixture at approximately every 4 bases, or with any of the strings of 3 - 7 adjacent nucleotides in the primer sub-region targeting a long gene segment, can achieve a sufficient reduction in specificity without losing primer function. Without limiting the present invention to any theory or mode of action, it has been determined that modifying with an N mixture at approximately every 4 bases, or with any of the strings of 3 - 7 adjacent nucleotides in the primer sub-region targeting a long gene segment, can achieve a sufficient reduction in specificity without losing primer function. Without limiting the present invention to any theory or mode of action, it has been determined that modifying with an N mixture at approximately every 4 bases, or with any of the strings of 3 - 7 adjacent nucleotides in the primer sub-region targeting a long gene segment, can achieve a sufficient reduction in specificity without losing primer function. Without limiting the present invention to any theory or mode of action, it has been determined that modifying with an N mixture at approximately every 4 bases, or with any of the strings of 3 - 7 adjacent nucleotides in the primer sub-region targeting a long gene segment, can achieve a sufficient reduction in specificity without losing primer function.

[0100] Promoting the interaction of the primers of the present invention with the target DNA can be carried out by any suitable method that may be more feasible. Such methods will be known to those skilled in the art. Methods for achieving primer-induced amplification are also very well known to those skilled in the art. In one method, the amplification is , polymerase chain reaction, NASBA, or strand displacement amplification. Preferably, the amplification is , polymerase chain reaction.

[0101] References to "sample" should be understood as references to biological or non-biological samples There are. Examples of non-biological samples include, for example, nucleic acid products of synthetically generated nucleic acid populations can be cited. References to "biological sample" refer to any sample of biological material derived from an animal that has been introduced into and subsequently removed from the body, including, but not limited to, cellular material, blood, mucus, feces, urine, tissue biopsy specimens, or body fluids (e.g., physiological saline extracted from the lungs after lung lavage or a solution taken from enema lavage), and should be understood as references to any sample of biological material derived from a mammal or mammalian tissue culture. Biological samples tested according to the methods of the present invention can be tested directly or may require some form of processing prior to testing. For example, biopsy specimens may require homogenization prior to testing . Additionally, biological samples may require the addition of reagents such as buffers to make the sample fluid, unless it is already in liquid form.

[0102] As long as the target DNA is present in the biological sample, the biological sample can be tested directly or, alternatively, all or part of the nucleic acid material present in the biological sample can be isolated prior to testing . Pretreatment of the target nucleic acid molecule prior to testing, for example, inactivation of live virus or ​ Migration on the gel is within the scope of the present invention. Also, the biological sample can be freshly collected, or may have been stored (e.g., by freezing) before the test, or may have been processed (e.g., by culturing) separately before the test and should be understood as such.

[0103] The reference to "contacting" the sample with the primer is to be understood as a reference to facilitating the mixing of the primer with the sample such that an interaction (e.g., hybridization) can occur. Means for achieving this purpose will be well known to those skilled in the art.

[0104] The selection of the most suitable type of sample for testing according to the methods disclosed herein will depend on the nature of the circumstances such as the nature of the condition being monitored. For example, in a preferred embodiment, a neoplastic condition is the subject of the analysis. If the neoplastic condition is lymphocytic leukemia, a blood sample, lymph fluid sample, or bone marrow aspirate is likely to provide a suitable test sample. If the neoplastic condition is lymphoma, a lymph node biopsy or a blood or bone marrow sample is likely to provide a suitable tissue source for the test. Consideration will also be required as to whether the original source of the neoplastic cells is being monitored, or whether the presence of metastases or other forms of spread of the neoplasm from the origin is to be monitored. In this regard, it may be desirable to collect and test several different samples from any one mammalian source. Selecting the appropriate sample for a given detection scenario will be within the skill of the art.

[0105] ​​​​​​As used herein, the term "mammal" within the scope of use includes humans, primates, domestic animals ( e.g., horses, cows, sheep, pigs, donkeys), laboratory test animals (e.g., mice, rats , rabbits, guinea pigs), companion animals (e.g., dogs, cats), and captive wild animals (e.g., kangaroos, deer, foxes), and preferably, the mammal is a human or a laboratory test animal. Even more preferably, the mammal is a human.

[0106] The method of this aspect of the present invention provides means for detecting the presence of a target nucleic acid region of interest (e.g., for diagnostic or monitoring purposes), and optionally both means for quantifying and / or isolating the target. Thus, means are provided for detecting, enriching, or purifying a nucleic acid population of a target of interest for any purpose, such as further analysis of the target.

[0107] Another aspect of the present invention provides a method for detecting and / or monitoring a clonal cell population of a mammal, wherein the clonal cell is characterized by an Ig or TCR nucleic acid region characterized by rearrangement of two or more V, D, or J gene segments, and the method includes the following. (i) Contacting the forward and reverse primers as described above with the DNA material of a biological sample derived from a mammal for a time and under conditions sufficient to promote the interaction between the primer and the target nucleic acid molecule. (ii) (a) An annealing temperature within the range between Tc and (Tc - 3°C) of the amplification reaction of interest, and / or (b) at least one primer containing at least one A and / or T nucleotide at the 3' terminal nucleotide position of the primer, and optionally, means for performing melting curve analysis, are used to amplify the nucleic acid target. ​ amplifying, and (iii) detecting the amplification product.

[0108] In one embodiment, the at least one primer is an ASO primer.

[0109] In another embodiment, the method is within the range between Tc and (Tc - 3°C) of the amplification reaction of the subject, annealing temperature, and using both primers having an A or T nucleotide at the position of the 3'-terminal nucleotide of the primer. including.

[0110] In yet another embodiment, the nucleic acid is DNA.

[0111] In yet another embodiment, each of the two 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the three 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the four 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the five 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the six 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, each of the six 3'-terminal nucleotide positions of the primer has an A and / or T nucleotide. In yet another embodiment, melting curve analysis is performed.

[0112] In yet another embodiment, the method preferably targets the recombinant Ig or TCR of interest

[0113] and The method includes a Ct determination using a probe of interest, which is bound to a detection means.

[0114] In a further embodiment, the amplification reaction is a polymerase chain reaction.

[0115] In yet another embodiment, the primer directed to a downstream gene segment is The target segment is

[0116] Reference to "cells" includes all forms of cells from any species, and variants or variants thereof. Preferably, the cells are lymphoid cells, but In particular, the methods of the present invention are directed to cells that may have undergone partial or complete Ig or TCR rearrangement. The present invention may be practiced with any type of cell that is capable of carrying out the method of the present invention. Without being limited to the mode of action, can cells constitute a living organism (in the case of unicellular organisms)? Or individual cells can be more or less specialized (differentiated) for specific functions. A living organism may be a subunit of a living organism. All living organisms are made up of one or more cells. The subject cells are part of a biological sample that is the subject of testing in a syngeneic, allogeneic, or xenogeneic context. Syngeneic situations can form part of clonal cell populations and the biological This means that the samples share the same MHC genotype. This would most likely be the case when screening for the presence of a neoplasm. An "allogeneic" situation occurs when the subject clone population is in fact identical to the individual from whom the biological sample was taken. This is the case when different MHCs are expressed. This is the case for conditions such as graft-versus-host disease. Relatedly, screening for the expansion of transplanted donor cell populations (e.g., immunocompetent bone marrow transplants) can occur when “Xenogeneic” situations are those where the subject clonal cells are of a species completely different from the subject from which the sample is derived. This can occur, for example,

[0117] when a potentially neoplastic donor population is derived from xenotransplantation. “Variant” of the subject cells includes cells that exhibit some, but not all, of the morphological or phenotypic characteristics or functional activities of the cells of which they are variants, but is not limited thereto. “Mutant” includes, but is not limited to,

[0118] cells that are modified, either naturally or non-naturally, such as cells that are genetically modified. In this regard, neoplastic cells that undergo further nuclear rearrangement or mutation to generate genetically distinct neoplastic cell populations are also different clonal cell populations, but are “clonal” cell populations. In another example, T or B lymphocytes that expand in response to acute or chronic infections or immune stimulation are also “clonal” populations of cells within the description provided herein. In yet another example, clonal cell populations are clonal microbial populations, such as drug-resistant clones that arise within a

[0119] larger microbial population. Preferably, the subject clonal cell population is a

[0120] neoplastic cell population or a clonal immune cell population. In one embodiment, the clonal cells are a population of clonal lymphoid cells.

[0120] References to “lymphoid cells” refer to immunoglobulin or TCR variable region gene segments It should be understood as a reference to any cell that is rearranging at least one germline set. The immunoglobulin variable regions encoding genomic DNA that can be rearranged include variable regions associated with the heavy chain or the κ or λ light chain, while the TCR chain variable regions encoding genomic DNA that can be rearranged include the α, β, γ, and δ chains. In this regard, a cell is understood to fall within the scope of the description of "lymphoid cells" if it is rearranging the DNA of at least one immunoglobulin or TCR gene segment region encoding a variable region. The cell need not also be transcribing and translating the rearranged DNA. In this regard, "lymphoid cells" include, but are not limited to, within their scope, immature T and B cells that are rearranging TCR or immunoglobulin variable region gene segments but have not yet expressed the rearranged chain (e.g., TCR thymocytes), or have not yet rearranged both chains of the TCR or immunoglobulin variable region gene segments. This definition further extends to lymphoid-like cells that have undergone at least some TCR or immunoglobulin variable region rearrangement, but which may not otherwise exhibit all of the phenotypic or functional characteristics conventionally associated with mature T or B cells. Thus, the methods of the present invention can be used, without limitation, to monitor the neogenesis of cells including lymphoid cells, activated lymphoid cells, or non-lymphoid / lymphoid-like cells at any stage of differentiation of development, provided that at least some rearrangement of the gene region of one variable region has occurred. It can also be used to monitor clonal expansion occurring in response to a specific antigen.

[0121] With respect to this aspect of the invention, reference to "monitoring" is to be understood as referring to testing a subject for the presence or level of a clonal cell population of the subject after an initial diagnosis of the presence of the population. It should be understood to refer to testing a subject for the presence or level of a clonal cell population of the subject after an initial diagnosis of the presence of the population. "Monitoring" includes reference to performing either a single isolated test or a series of tests over days, weeks, months, or years. The tests may be performed for any number of reasons, including but not limited to, to assist in making decisions regarding appropriate treatment, or to test new forms of treatment, such as predicting the likelihood of recurrence of a mammal in remission, screening for minimal residual disease, monitoring the effectiveness of a treatment protocol, checking the status of a patient in remission, monitoring the progression of the status before or after application of a treatment regimen. Accordingly, the methods of the invention are useful both as clinical and research tools. It is preferred that the rearrangement of at least one variable region gene region be complete, but nevertheless, it should be understood that the methods of the invention are applicable to the monitoring of neoplastic cells exhibiting only partial rearrangement. For example, B cells that have undergone only a DJ rearrangement event are cells that have undergone only a partial rearrangement. A complete rearrangement will not be achieved until the DJ rearrangement segment further rearranges with a V segment.

[0122] Accordingly, the methods of the invention can be designed to detect partial or complete variable region rearrangements of one TCR or immunoglobulin chain using a reference molecule complementary to this marker sequence, or, for example, in cases where higher specificity is required and the neoplastic cells are rearranging both variable regions of both the TCR or immunoglobulin chains, probe molecules directed to both forms of rearrangement can be utilized. It is preferred that the rearrangement of at least one variable region gene region be complete, but nevertheless, it should be understood that the methods of the invention are applicable to the monitoring of neoplastic cells exhibiting only partial rearrangement. For example, B cells that have undergone only a DJ rearrangement event are cells that have undergone only a partial rearrangement. A complete rearrangement will not be achieved until the DJ rearrangement segment further rearranges with a V segment. Accordingly, the methods of the invention can be designed to detect partial or complete variable region rearrangements of one TCR or immunoglobulin chain using a reference molecule complementary to this marker sequence, or, for example, in cases where higher specificity is required and the neoplastic cells are rearranging both variable regions of both the TCR or immunoglobulin chains, probe molecules directed to both forms of rearrangement can be utilized. or, for example, in cases where higher specificity is required and the neoplastic cells are rearranging both variable regions of both the TCR or immunoglobulin chains, probe molecules directed to both forms of rearrangement can be utilized.

[0123] References to "neoplastic cells" should be understood as references to cells showing abnormal "growth". The term "growth" should be understood in its broadest sense and includes reference to proliferation. In this regard, an example of abnormal cell growth is uncontrolled proliferation of cells. Uncontrolled proliferation of lymphoid cells can result in a cell population that takes either the form of a solid tumor or a single cell suspension (such as that observed in the blood of a leukemia patient). Neoplastic cells can be either benign or malignant cells. In a preferred embodiment, the neoplastic cells are malignant cells. In this regard, references to "neoplastic conditions" are references to the presence of neoplastic cells in a subject mammal. References to "neoplastic lymphoid conditions" include references to conditions characterized by the presence of an abnormally large number of neoplastic cells occurring in leukemia, lymphoma, and myeloma, although this manifestation also includes references to situations where the number of neoplastic cells present in a mammal is below a threshold that is considered to distinguish between a transition to a remission state or the reverse from an overt disease state in the mammal (the number of cells present during remission is often referred to as "minimal residual disease"). Further, even if the number of neoplastic cells present in a mammal is below the threshold detectable by screening methods utilized prior to the emergence of the present invention, the mammal is considered to exhibit a "neoplastic condition". In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states.

[0124] In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states. In this regard, disease states suitable for analysis are any lymphoid malignancies such as acute lymphoblastic leukemia, chronic lymphocytic leukemia, lymphoma, and myeloma. Monitoring of minimal residual disease is important in all of these states.

[0125] Preferably, the condition is a neoplasm, and even more preferably, a lymphoid neoplasm .

[0126] In certain embodiments, the methods of the invention are used to detect minimal residual disease in the context of lymphocytic leukemia .

[0127] Yet another aspect of the invention relates to isolated primers as described above

[0128] Yet another aspect of the invention relates to kits for facilitating the identification of Ig or TCR nucleic acid regions characterized by rearrangement of two or more V, D, or J gene segments, the kit comprising compartments suitable for containing any one or more of oligonucleotide primers as defined above, reagents useful for facilitating interaction of the primers with a target nucleic acid molecule, and reagents useful for enabling such interaction to effect amplification of the nucleic acid target . For example, additional compartments may be included that receive biological or non-biological samples . . .

[0129] Further features of the invention are more fully described in the following non-limiting examples

Example

[0130] Example 1 Guidelines for Primer Design 3' End - The last 3' base is A / T - Preferably, the last 2 or 3 bases are A / T - A / T may be present as a base for the last up to 4 most 3' bases. In more cases, there is a risk that the primer will not function efficiently - At least 2 G / C in the last 7 bases

[0131] Design when using two N regions - The primer targets some or all of N1, all of D, and some or all of N2 to be. - It may be advantageous to include up to 4 bases at the 5' of N1 and / or 1 - 3 bases at the 3' of N2. This utilizes the semi - native V - N1 and N2 - J junctions, as well as any A / T bases provided at J - If the Tm of the primer is too high, above 74°C, one or more N bases can be inserted into the D region to lower the effective Tm

[0132] Design when using one N region The only N region may be available in the presence of V - N - J or partial D - N - J rearrangement. Optionally, even if two N regions are available, especially if they contain a long, preferably non - uniform base sequence and enable a good 3' - end design, only one may be used It may be determined that way. When the N region is short and / or it is difficult to design a primer with a good 3' - end - When the D region follows the N region, especially if this improves the 3' - end, it is usually advantageous to extend the primer by 4 - 6 bases into the D region. This utilizes the semi - native N - D junction - When the J region follows the N region, optionally when A / T is investigated at the 3' - end, it is usually advantageous to extend the primer by 1 - 3 bases into the J region. However, especially in this situation, it is important to test several different primers - When there is a 3' - end of the V region before the N region, it may be advantageous to incorporate 3 - 5 bases of V at the 5' - end, as long as the primer does not become too long. This utilizes the semi - native V - N junction ​​​​​​​​​​​is used.

[0133] Tm and annealing temperature The length and Tm of the ASO primer depend on the nature of the rearrangement, but in many cases, there is some flexibility in determining the exact length and Tm. In that case, it is recommended to design the primer such that the Tm is approximately the same as or 1 °C lower than the Tm of the reverse primer is recommended. The annealing temperature used in PCR depends on the Tm of the primer. The guidelines are Tm = 59 - 61: annealing temperature = 63 - 64, Tm = 69 - 71: annealing temperature = 72, Tm = 71 - 74: annealing temperature = 75, Tm > 74: annealing temperature = 75, but consider the use of N bases to lower the Tm is considered. These guidelines usually result in an annealing temperature within 3 °C of the Tc value. However, especially when the N region contains 5 or fewer bases, some difficulty may be expected for the rearrangement primer using the only N region. In such rearrangements, the annealing temperature should be near or at the Tc value so as to produce a minimum of non-specificity or no non-specificity at all. It is also advisable to synthesize and test several candidate primers is advisable.

[0134] Note The above are only guidelines and should depend on the temperature gradient results and primer sequences. In some patients, it may be desirable to synthesize and test multiple candidate primers.

[0135] Example 2 Various sets of J primers used for IGH rearrangement ​​​​The normal set used is either AAM or setB.

Table 2

[0136] Example 3 Primer test 1. Determination of amplification efficiency, Tc, and specificity One or several primers are synthesized for each patient. Using a gradient of annealing temperature each primer is tested for its ability to amplify from leukemia DNA and failure of amplification from non-leukemia DNA. Examples are as follows.

Table 3

[0137] 2. Next, the selected primers are tested to confirm that their ability to amplify the rearranged target gene of leukemia DNA is not inhibited by the co-presence of non-leukemia DNA. (no data).

[0138] An example of the test is shown below. The presence of 500 ng or 1 μg of non-leukemia DNA did not result in a change in the CT observed with 40 0 pg of leukemia DNA. Amplification was carried out with 500 ng ​​of non-leukemic DNA alone or in the water control. [Table 4]

[0139] 3. Final Test for Non-specificity This can be done at the end of primer post-processing or during the first final experiment where MRD is measured. The primers are tested on 20 wells, each well containing contains 1 μg of pooled DNA from five non-leukemic individuals.

[0140] Example 4 Patient ASO primer sequences and Tm and Tc values V or V, N, D, N, J indicate the rearrangement region targeted by the primer. The assays were for samples from patients with acute lymphoblastic or chronic lymphocytic leukemia. [Table 5]

[0141] Example 5 For testing gradient primers Each primer is probed in duplicate at six different temperatures. Normal DNA is run at two low temperatures to check the primers for non-specificity. will be done. Dilute diagnostic sample to 200 pg / μl. Add 2 μl / tube and 28 μl / plate. Requires an immerset Dilute normal DNA to 250ng / μl Add D0 DNA to columns A, B, C, D, E, and F. Add PBL DNA to rows G and H The table is for six primers, but it is possible to use fewer primers on a plate or strip. can be reduced to perform

Table 6

[0142] Example 6 Appendix Table 5. Worksheet 4: Configure the mixture for quantification using a single primer Patient diagnostic DNA at 1 ng / μl Pbl DNA requires 120 μl at 250 ng / μl, 4 μl / tube. Dilution of the MR tube From 1 ng / μl to 3 μl + 27 μl of FG3 at 0.1 ng / μl From 0.1 ng / μl to 2 μl + 18 μl of FG3 at 0.01 ng / μl ​​​​250 ng / μl of pbl is required to make up the mixture added at 20 μl / tube. Required. [Table 7]

[0143] Example 7 Quantification using a single primer for two samples, two tubes, and five tubes Quantification [Table 8] Execution: 91°C, 3 minutes 97°C, 15 seconds; 72°C, 30 seconds × 5 cycles, 96°C, 15 seconds; 72°C, 30 seconds × 5 cycles, 94°C, 15 seconds; 72°C, 30 seconds × 35 cycles

[0144] Example 8 Melting curve analysis PCR contains the fluorescent dye Syto82 at a concentration of 0.5 μM. Preliminary tests showed that this concentration did not extend the Ct as measured by the reporter probe. Fluorescence was measured as the temperature increased gradually, and the results were analyzed using an appropriate program. The first derivative of the fluorescence vs. temperature curve shows peaks for non-specific and specific amplification. As the temperature rises gradually, fluorescence is measured, and the results are analyzed using an appropriate program. The first derivative of the fluorescence vs. temperature curve shows peaks for non-specific and specific amplification.

[0145] Those skilled in the art will understand that the invention described herein permits changes and modifications other than those specifically described. The invention is to be understood to include all such variations and modifications. The invention also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or shown herein, as well as any and all combinations of any two or more of said steps or features. Those skilled in the art will understand that the invention described herein permits changes and modifications other than those specifically described. The invention is to be understood to include all such variations and modifications. The invention also includes, individually or collectively, all of the steps, features, compositions, and compounds referred to or shown herein, as well as any and all combinations of any two or more of said steps or features.

[0146] List of references Brisco, M.J., Bartley, P.A., and Morleyh A.A. Antisense PCR: A simple and robust method for performing nested single-tube PCR.A nalytical Biochemistry 2011;409:176-182 Bruggemann M, van der Velden VH, Raff T, Dr oese J, Ritgen M, Pott C, et al. Rearranged T-cell receptor beta genes represent pow erful targets for quantification of mini mal residual disease in childhood and ad ult T-cell acute lymphoblastic leukemia. Leukemia.2004;18(4):709-19. Li A-H, Forestier E, Rosenquist R, and Roos G. Minimal residual disease quantificati on in childhood acute lymphoblastic leuk emia by real-time polymerase chain react ion using the SYBR green dye. Experimenta l Hematology 2002;30:1170-1177 Morley AA, Latham S, Brisco MJ, Sykes PJ, Su tton R,Hughes E,et al.Sensitive and spec ific measurement of minimal residual dis ease in acute lymphoblastic leukemia.J M ol Diagn.2009;11(3):201-10. Nakao M,Janssen JW,Flohr T,Bartram CR.Ra pid and reliable quantification of minim al residual disease in acute lymphoblast ic leukemia using rearranged immunoglobu lin and T-cell receptor loci by LightCyc ler technology.Cancer Res.2000;60(12):32 81-9. Pongers-Willemse MJ,Seriu T,Stolz F,d’An iello E,Gameiro P,Pisa P,et al.Primers a nd protocols for standardized detection of minimal residual disease in acute lym phoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: repo rt of the BIOMED-1 CONCERTED ACTION:inve stigation of minimal residual disease in acute leukemia.Leukemia.1999;13(1):110- 8. van der Velden VH,Boeckx N,van Wering ER ,van Dongen JJ.Detection of minimal resi dual disease in acute leukemia.J Biol Re gul Homeost Agents.2004;18(2):146-54. van der Velden VH,Panzer-Grumayer ER,Caz zaniga G,Flohr T,Sutton R,Schrauder A,et al.Optimization of PCR-based minimal re sidual disease diagnoses for childhood acute lymphoblastic leukemia in a multi -center setting.Leukemia.2007;21(4):706- 13. van der Velden VH,van Dongen JJ.MRD dete ction in acute lymphoblastic leukemia pa tents using Ig / TCR gene rearrangements as targets for real-time quantitative PC R.Methods Mol Biol.2009;538:115-50. van der Velden VH, Wijkhuijs JM, Jacobs DC ,van Wering ER,van Dongen JJ.T cell rece ptor gamma gene rearrangements as target s for detection of minimal residual disease ase in acute lymphoblastic leukemia by r eal-time quantitative PCR analysis.Leuke mia.2002;16(7):1372-80. van der Velden VH,Willemse MJ,van der Sc hoot CE,Hahlen K,van Wering ER,van Donge n JJ.Immunoglobulin kappa deleting element nt rearrangements in precursor-B acute l ymphoblastic leukemia are stable targets for detection of minimal residual disea se by real-time quantitative PCR.Leukemia a.2002;16(5):928-36. van der Velden VHJ,Noordijk R,Brussee M, Hoogeveen P,Homburg C,de Haas V,C.van de r Schoot E,van Dongen JJM.Minimum residue al disease diagnostics in acute lymphobl astic leukemia:Impact of primer charact eristics and size of junctional regions. British Journal of Haematology,2014,164, 451-464 Verhagen OJ,Willemse MJ,Breunis WB,Wijkh uijs AJ,Jacobs DC,Joosten SA,et al.Appli cation of germline IGH probes in real-ti me quantitative PCR for the detection of minimal residual disease in acute lymph oblastic leukemia.Leukemia.2000;14(8):14 26-35.

Claims

1. Ig or TCR characterized by rearrangements of two or more V, D, or J gene segments 1. A method for amplifying a nucleic acid region, the method comprising: Forward and reverse primers directed to the rearranged Ig or TCR nucleic acid region contacting a nucleic acid sample of interest with the (i) an annealing temperature in the range between the Tc and (Tc-3°C) of the subject amplification reaction; and / or (ii) at least one A and / or T nucleotide at the 3'-terminal nucleotide position At least one primer comprising and, optionally, means for performing melting curve analysis. amplifying said nucleic acid sample using The method comprising:

2. A method for detecting and / or monitoring a clonal cell population in a mammal, comprising: The cells express Ig or IgE markers characterized by rearrangements of two or more V, D, or J gene segments. The method is characterized by a TCR nucleic acid region, the method comprising: (i) the forward and reverse nucleic acid sequences directed to the rearranged Ig or TCR nucleic acid region; a primer and a DNA material of a biological sample derived from a mammal, said target nucleic acid molecule being identified; contacting under conditions sufficient to promote interaction of said primers; (ii)(a) an annealing reaction within the range between the Tc and (Tc-3° C.) of the subject amplification reaction; and / or (b) at least one primer, At the 3' terminal nucleotide position, contains at least one A and / or T nucleotide , said primers, and, optionally, means for performing a melting curve analysis. amplifying the acid target; and (iii) detecting the amplification product. The method comprising:

3. The method further comprising: an annealing temperature within the range between the Tc and (Tc-3°C) of the subject amplification reaction; One or more primers, comprising at the 3′ terminal nucleotide position of said primer, A or a T nucleotide.

3. The method of claim 1 or 2, comprising using both

4. The method according to any one of claims 1 to 3, wherein the nucleic acid region is DNA.

5. At each of the two 3' most terminal nucleotide positions of said primer, A and / or T The method according to any one of claims 1 to 5, wherein a nucleotide is present.

6. At each of the three most 3' nucleotide positions of said primer, an A and / or a T The method according to any one of claims 1 to 5, wherein a nucleotide is present.

7. At each of the four 3' most terminal nucleotide positions of said primer, A and / or T The method according to any one of claims 1 to 5, wherein a nucleotide is present.

8. At each of the five most 3' nucleotide positions of said primer, an A and / or a T The method according to any one of claims 1 to 5, wherein a nucleotide is present.

9. At each of the six most 3' nucleotide positions of said primer, an A and / or a T The method according to any one of claims 1 to 5, wherein a nucleotide is present.

10. Either or both of the forward and reverse primers are is designed to contain a T nucleotide, method.

11. Only the reverse primer is designed to contain the A and / or T nucleotides. The method according to any one or more of claims 1 to 10,

12. The VDJ rearrangement is a partial rearrangement, the rearranged V and D gene segments, The rearranged D and J gene segments, or the rearranged V and J gene segments Any one of claims 1 to 11, wherein there is an N region located at the junction between one or more of The method according to any one of the preceding claims.

13. the VDJ rearrangement is a complete rearrangement, the rearranged V and D gene segments, of the rearranged D and J gene segments, or the rearranged V and J gene segments Any one of claims 1 to 11, wherein there is an N region located at the junction between one or more The method described above.

14. The VDJ rearrangement is a partial rearrangement, and the V gene segment, the D gene segment, In one embodiment, the N region is located in one or more of the V gene segments.

13. The method according to any one or more of claims 1 to 12.

15. The VDJ rearrangement is a complete rearrangement, and the V gene segment, the D gene segment, In one embodiment, the N region is located in one or more of the V gene segments.

14. The method according to any one or more of claims 1 to 11 or 13.

16. At least one of the primers is a primer for one of the rearranged V, D, and J gene segments. 1, comprising hybridization subregions directed to N gene segments.

15. The method according to any one or more of claims 1 to 14.

17. At least one of the primers is a primer for detecting at least one of the rearranged V, D, and J gene segments. Contains hybridization subregions covering at least two N gene segments The method according to any one or more of claims 1 to 14.

18. The primer directed to the J gene segment has at least one The method according to any one of claims 1 to 17, comprising A and / or T nucleotides of the formula:

19. A primer subregion is a mixture of nucleotides from the N mixture and nucleotides from the subregion. The method according to any one of claims 1 to 18, wherein the amino acid sequence of method.

20. 20. The method of claim 19, wherein every fourth nucleotide of the subregion is substituted.

21. The method according to claim 20, wherein a sequence of 3 to 7 adjacent nucleotides of the subregion is replaced. method.

22. The method according to any one of claims 1 to 21, wherein a melting curve analysis is performed.

23. 23. The method of claim 22, wherein the melting curve analysis is performed using a dye.

24. 24. The method of claim 23, wherein the dye is a fluorophore.

25. The method according to any one of claims 1 to 26, wherein the method comprises a Ct determination step.

26. The Ct determination is performed using a probe directed to the rearranged Ig or TCR region.

26. The method of claim 25, wherein the probe is attached to a detection means.

27. 27. The method of claim 26, wherein the detection means is a fluorescent molecule.

28. The method according to any one of claims 1 to 27, wherein the at least one primer is an ASO primer.

13. The method according to any one of claims 1 to 12.

29. The primer directed to the downstream gene segment is directed to the J segment. The method according to any one of claims 1 to 28,

30. The method according to any one of claims 1 to 29, wherein the amplification is a polymerase chain reaction. Law.

31. 2. The method of claim 1, wherein the condition is a neoplasm, and even more preferably a lymphoid neoplasm. The method according to

32. 32. The method of claim 31, wherein the lymphoid neoplasm is a lymphoid malignancy.

33. The lymphoid malignancies include acute lymphoblastic leukemia, chronic lymphocytic leukemia, and myeloma.

33. The method of claim 32, wherein:

34. The method of claim 2, wherein the method is used to detect minimal residual disease.

35. 35. The method of claim 34, wherein the minimal residual disease is detected in association with lymphocytic leukemia. 。

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