Method and composition for multi-step primer extension reaction

Abstract

Methods and compositions for multi-step primer extension reactions, such as multiplex polymerase chain reaction (PCR) and reverse transcriptase PCR, are described. [Solution] The primer extension steps are performed in a closed container without opening the container between steps. The multi-step primer extension method and composition utilizes an earlier stage primer in an earlier stage and a later stage primer in a later stage, where the later stage primer is blocked from extension during the earlier stage. The blocked primers of this technology contain photocleavable protecting groups that are substantially inactive until the protecting group is cleaved by exposure to ultraviolet light. The blocked primers can be activated by ultraviolet light without opening the container.

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 994,989, filed on 26 March 2020, the contents of which U.S. Provisional Patent Application No. 62 / 994,989 are incorporated in their entirety by reference.

[0002] This disclosure relates to methods and compositions for multi-step primer extension reactions such as multiplex polymerase chain reaction (PCR) and reverse transcriptase-PCR. [Background technology]

[0003] Polymerase chain reaction (PCR) is a specific amplification method for DNA sequences. PCR is a useful and widely applied method for DNA target amplification in next-generation sequencing (NGS) library preparation. Specifically, primers hybridize to their target sequence in a mixture of nucleic acids and are extended, followed by further rounds of primer hybridization and extension. PCR allows for exponential amplification of sequences between primers, making it a highly sensitive technique. However, PCR can lead to unwanted amplification products. Firstly, if a primer binds to other sequences in addition to its target, off-target amplification can reduce the yield of the target sequence. Secondly, PCR primers need to be at a much higher concentration than the target sequence (to support later rounds of the exponential reaction), and therefore, primers can sometimes react with other primers to produce primer dimers. Thirdly, different target sequences may amplify with different efficiencies depending on their length, GC content, primer sequence, etc.

[0004] Multiplex PCR (mPCR) is a process in which many (sometimes hundreds or thousands) primers are used in the extension reaction. This is convenient because many target sequences can be amplified in the same tube, and in some cases, many target sequences can be amplified from the same aliquot of the sample. However, the problems of mistargeting amplification, primer dimer formation, and heterogeneous amplification combine in mPCR. In fact, the primer dimer problem can be rapidly exacerbated because each new pair of primers added to the multiplex reaction can potentially interact with all the other primers in the mixture.

[0005] Multiplex PCR has the potential to significantly save time and effort in the laboratory. This technique has been applied to many areas of human DNA testing, including gene deletion analysis, mutation and polymorphism analysis, quantitative analysis, and reverse transcription (RT)-PCR. In the field of infectious diseases, multiplex PCR has been used for the identification of viruses, bacteria, and parasites. However, the use of mPCR presents several problems, including low sensitivity, low specificity, preferential amplification of certain specific targets, and / or amplification of unintended sequences.

[0006] Typically, multiplex PCR involves two separate steps (i.e., multi-step PCR). In the first step, a target-specific polynucleotide is amplified using a target-specific primer with a universal sequence, and the universal forward and reverse sequences are added to each amplicon. The product is then purified before the subsequent PCR amplification reaction to remove unreacted target-specific primers and other reagents. In the subsequent PCR reaction, a universal primer designed to hybridize to the universal sequence is used to amplify the amplicons from the previous step and add any further sequences required for further processing and identification purposes (such as adapters).

[0007] This system is labor-intensive. Furthermore, competition between target-specific primers and universal primers can result in biased amplification, requiring the subsequent reaction mixture to be stopped and purified. This can introduce errors and contamination into the system. Therefore, it would be desirable to find a way to carry out both steps in a single reaction vessel. Moreover, it would be particularly preferable (in terms of preventing contamination) and convenient if two or more steps could be carried out without opening the reaction vessel.

[0008] In reverse transcriptase polymerase chain reaction (RT-PCR), RNA is reverse transcribed into cDNA in the first step, and then the cDNA is amplified in subsequent steps, usually by target-specific primers, in the PCR step. RT-PCR has the same problems as multi-step PCR. This is because gene-specific primers are amplified at relatively low temperatures (37-60°C). oIt is because it is non-specifically primed during cDNA synthesis carried out in (C). Specificity is generally improved by performing reverse transcription and PCR in two separate containers. Thereby, it is prevented that PCR primers interact with each other or with RT primers such as oligo(dT), random hexamers or gene-specific reverse primers that are used to initiate cDNA synthesis. However, opening the tube between the RT step and the PCR step increases the labor and the risk of contamination.

Prior Art Documents

Patent Documents

[0009]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Summary of the Invention

Means for Solving the Problems

[0011] The present technology relates to a novel method for performing a multi-step polymerase chain reaction in a closed container, wherein the mixture comprises: i) a polynucleotide target, ii) a primer extendable in the forward stage, iii) a primer in the backward stage having a photocleavable protecting group at its 3'-end, iv) a primer extension enzyme, and v) optionally other reagents as desired. Examples of primers in the forward stage include target-specific primers and reverse transcriptase (RT) primers. The target-specific primer may have a 5'-region and a 3'-region, the 3'-region having a target-specific sequence and the 5'-region having a universal sequence. When such a target-specific primer is used in the forward primer extension reaction, the primer in the backward stage may be a universal sequence having the universal sequence or a part thereof and the photocleavable protecting group at its 3'-end. In some embodiments, the container is closed after the preparation of the mixture, and the forward polymerase chain reaction is performed with the mixture to generate a target amplicon. The universal primer is unblocked in the mixture, generating an unblocked universal primer having the universal sequence or a part thereof. In some embodiments, the unblocking step is performed without opening the container. The backward primer extension reaction is performed with the unblocked primer and the target amplicon, wherein the unblocked primer amplifies the target amplicon. In some embodiments, the unblocking step is performed by exposing the universal primer to ultraviolet light in the closed container.

[0012] In another aspect, the technology relates to a novel method for performing multi-step PCR, wherein the composition comprises a) a polynucleotide target, b) a primer-extendable pre-primer, and c) a post-primer having a photocleavable protecting group at its 3' end. Examples of pre-primers include target-specific primers and reverse transcriptase (RT) primers. The target-specific primer may have a 5' region and a 3' region, where the 3' region contains a target-specific sequence and the 5' region contains a universal sequence. If such a target-specific primer is the pre-primer, the post-primer may be a universal sequence having the universal sequence or a portion thereof and a photocleavable protecting group at its 3' end. In some embodiments, the composition is housed in a container that is closed after the composition is prepared. Exposure to ultraviolet light can unblock the post-primer and activate it for PCR amplification.

[0013] In another aspect, the technology relates to a novel method for performing multi-step RT-PCR in a closed container, wherein the mixture comprises i) a polynucleotide (RNA) target, ii) a front primer comprising an oligo(dT) primer, a random primer, or a target-specific RT primer for cDNA synthesis, iii) a back primer comprising a target-specific sequence at the 5' end and a photocleavable protecting group at the 3' end, iv) a reverse transcriptase, v) a DNA polymerase, and vi) any other reagents as desired. In some embodiments, the container is closed after the preparation of the mixture and heated to a constant temperature (37-60°C) before PCR thermal cycling. o Reverse transcription is performed in C). The target-specific PCR primers are unblocked without opening the container, and the PCR step is performed with the unblocked PCR primers and cDNA, during which the unblocked primers amplify one or more target amplicons. In some embodiments, the unblocking step is performed by exposing the blocked target-specific PCR primers to ultraviolet light in a closed container.

[0014] In another aspect, this technology relates to a novel composition for performing multi-step RT-PCR in a closed container. The composition comprises a pre-primer comprising a) a polyribonucleotide (RNA) target and b) an unblocked RT primer for cDNA synthesis, and a post-primer comprising a blocked primer for the post-primer extension reaction, wherein the post-primer has a photocleavable protecting group at its 3' end. The blocked post-primer may be a target-specific PCR primer, a random primer, or a universal primer. The post-primer can be unblocked after cDNA synthesis and activated for PCR amplification by exposure to ultraviolet light. In some embodiments, the composition is housed in a container that is closed after the composition is prepared.

[0015] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended in any way to limit the scope of this instruction. [Brief explanation of the drawing]

[0016] [Figure 1] A schematic diagram of an embodiment of the multi-step PCR primer, method, and composition is shown. [Figure 2] A reverse-phase HPLC trace is shown demonstrating the formation of a blocked universal primer with a photocleavage-type protecting group at its 3' end. [Figure 3] A schematic diagram of an embodiment of the multi-step RT-PCR primer, method, and composition is shown. [Figure 4] The reverse-phase HPLC trace demonstrates the deblocking of the blocked universal primer in the PCR tube by exposing the primer to 365 nm ultraviolet light for 10 seconds. [Figure 5]The image from the Bioanalyzer 2100 demonstrates that a photocleavable blocked primer was extended in PCR only after exposure of the primer to ultraviolet light. [Figure 6A] The bioanalyzer images show a single-container RT-PCR reaction performed with an unblocked RT primer in the preceding step, in the presence of a blocked subsequent primer. The blocked subsequent primer was unblocked for use in the subsequent step. [Figure 6B] The bioanalyzer images show a single-container RT-PCR reaction performed with an unblocked RT primer in the preceding step, in the presence of a blocked subsequent primer. The blocked subsequent primer was unblocked for use in the subsequent step. [Modes for carrying out the invention]

[0017] This technology relates to multi-step primer extension reactions, such as multiplex PCR and RT-PCR, using an unblocked pre-primer and a blocked post-primer. The blocked primer contains a photocleavable protecting group at its 3' end. A polynucleotide target is subjected to a primer extension reaction in the pre-step to form a product such as a target amplicon or target cDNA. For example, genomic DNA can be amplified in the pre-step using a target-specific primer having a target-specific sequence in the 3' region and a universal sequence in the 5' region. This pre-step product comprises a target amplicon with universal sequences at the 5' and 3' ends of the amplicon. The target amplicon is then amplified in the post-step primer extension reaction using a universal primer that has been unblocked by photocleavage of the protecting group, such as by exposure to ultraviolet light. As another example, an RNA target can be subjected to primer extension in the pre-step to generate target cDNA. The target cDNA can then be subjected to primer extension in the post-step using an unblocked target-specific primer or a universal primer.

[0018] Figure 1 shows a schematic diagram of the multi-step PCR method of this technology. The illustrated PCR method can be performed in a closed container containing a polynucleotide target, a target-specific primer having a universal sequence, and a 3'-blocked photocleavable universal primer having a photocleavable protecting group at its 3' end.

[0019] Figure 1 shows the amplification of a polynucleotide target in the preceding PCR using target-specific forward and reverse primers. The forward primer has a target-specific sequence, indicated as tag 1, which hybridizes to the target and a universal sequence, and the reverse primer also has a target-specific sequence, indicated as tag 2, which hybridizes to the target and another universal sequence. Amplification of the polynucleotide target in the preceding PCR generates a target amplicon that contains the polynucleotide target sequence and further has universal sequence tags 1 and 2 (or their complements) at the 5' and 3' ends of the target amplicon, respectively. The preceding PCR amplification is typically multiplex PCR amplification, in which multiple targets are amplified in parallel using multiple sets of target-specific primers. Exemplary sets of target-specific primers are available through Agilent's SureMASTR technology, such as in the BRCA MASTR DX assay.

[0020] Figure 1 also shows the subsequent amplification of a target amplicon with a set of 3'-blocked photocleavage-type universal primers. In this technique, the forward universal primer shown in Figure 1 includes a photocleavage-type protecting group at its 3' end, a 3' region containing the sequence of universal sequence tag 1 or a portion thereof, and an adapter sequence in its 5' region, indicated as adapter 1. The reverse universal primer shown in Figure 1 includes a photocleavage-type protecting group at its 3' end, a 3' region containing the sequence of universal sequence tag 2 or a portion thereof, a molecular identifier indicated as MID 1, and an adapter sequence in its 5' region, indicated as adapter 2. The photocleavage-type protecting groups in the forward and reverse universal primers are indicated by the label "cancelled". The blocked universal primers are present in the reaction mixture during the preceding PCR, but are substantially inactive for PCR amplification until they are unblocked. The blocked universal primers can be unblocked and activated for subsequent PCR amplification by exposure to ultraviolet light. After exposure to ultraviolet light, the unblocked universal primers are active for PCR amplification. As shown in Figure 1, the subsequent PCR amplification results in universal amplification of the previous amplicon, generating the subsequent amplicon. Therefore, the universally amplified subsequent amplicon contains target-specific primer and universal primer sequences (e.g., adapter 1 and adapter 2 sequences).

[0021] The blocked universal primers in this technology can be unblocked by exposure to ultraviolet light, allowing them to be added after the PCR step. Therefore, the universal primers can be present in the mPCR reaction mixture during the initial target amplification without interfering with it. Remarkably, the blocked universal primers are substantially inactive before the PCR step but become activated for the subsequent PCR step. Furthermore, the universal primers can be unblocked and activated by the photocleavage of their protecting groups by exposing the entire PCR mixture container to ultraviolet light. This unblocking capability is convenient and extremely beneficial because it reduces or avoids contamination, as the subsequent PCR can be performed without opening the PCR mixture container and adding the universal primers after the initial PCR step.

[0022] For example, after the pre-PCR stage is complete, 3'-blocked photocleaved universal primers can be deblocked by exposure to ultraviolet light, such as 365 nm light. Exposure to ultraviolet light can be carried out in any suitable manner. Deblocking may be done, for example, by removing the PCR vessel from the thermocycler and placing it on an ultraviolet light source such as an ultraviolet light box. Alternatively, the PCR thermocycler may be modified to directly apply UV light. In some cases, the universal primers may be deblocked by applying ultraviolet light while they are still in the same closed PCR reaction vessel. The same closed PCR reaction vessel can then proceed to the subsequent amplification stage with the now deblocked universal primers.

[0023] In another aspect, this technology relates to a multi-step RT-PCR in which the subsequent primers are target-specific primers containing a photocleavage-type protecting group at their 3' end. A polyribonucleotide target is reverse transcribed in the first step using reverse transcriptase and unblocked RT primers such as oligo(dT) primers, random primers, or target-specific reverse primers. The product of the preceding primer extension reaction is the target cDNA. The target cDNA can be amplified during the subsequent PCR using subsequent primers, such as target-specific primers that are unblocked by photocleavage of the protecting group.

[0024] Figure 3 shows a schematic diagram of the multi-step RT-PCR of this technology. The illustrated RT-PCR can be performed in a closed container containing a polyribonucleotide target, an unblocked RT primer, and a target-specific primer with a 3' photocleavage protecting group. The photocleavage protecting group in the target-specific primer is indicated by the label "cancelled". Figure 3 shows the reverse transcription of the polyribonucleotide target preceding the blocked target-specific primer using an unblocked reverse primer. Figure 3 also shows the amplification of the target amplicon following the removal of the 3' protecting group by exposure to ultraviolet light.

[0025] In some embodiments, the photocleavage protecting group of the technology is connected to a suitable reporter, such as a fluorophore. In other embodiments, the photocleavage protecting group of the technology is not connected to a reporter. It is possible that the photocleavage protecting group of the technology functions properly whether or not it contains a fluorophore, and that only the presence of the photocleavage protecting group is required.

[0026] Furthermore, since the photocleavage-type protecting group of this technology is removed by exposure to ultraviolet light, this protecting group does not affect the function of the primer elongation enzyme. Therefore, the photocleavage-type protecting group of this technology is suitable for use with standard PCR and RT-PCR components such as polymerase enzymes, nucleotides (dNTPs), and buffers.

[0027] Before describing the exemplary embodiments in more detail, the following definitions and explanations are provided to illustrate and define the meaning and scope of terms used in this description.

[0028] Numerical ranges include the numbers that define the range. Unless otherwise specified, nucleic acids are written from left to right in the 5' to 3' direction, and amino acid sequences are written from left to right in the amino to carboxyl direction.

[0029] This technology may employ techniques and descriptions from organic chemistry, polymer technology, molecular biology (including recombination technology), cell biology, biochemistry, and immunology, unless otherwise specified, and these are within the scope of such technologies. Such techniques include polymer array synthesis, hybridization, ligation, and detection of hybridization using labeling.

[0030] As used herein, singular forms such as “one” and “above” include multiple subjects unless the context clearly indicates otherwise. For example, the term “one primer” includes one or more primers, i.e., a single primer and multiple primers. “Multiple” includes at least two elements. In some cases, the plural is at least 10, at least 100, at least 100, at least 10,000, at least 100,000, at least 10 6 , at least 10 7 , at least 10 8 , or at least 10 9 Or, it may include more elements.

[0031] It should be further noted that claims may be written to exclude any of the elements. In this way, this wording is intended to serve as an antecedent basis for the use of exclusive terms such as "only" or "only" in relation to the detailing of elements of a claim or the use of "negative" limitations.

[0032] When used in the specification and the appended claims, and in addition to its ordinary meaning, the terms "substantial" or "substantially" mean within the limits or degrees acceptable to those skilled in the art. For example, "substantially inert" means that those skilled in the art consider the level of activity to be negligible.

[0033] The term "sample" as used herein relates to a material or mixture of materials containing one or more polynucleotides or fragments of interest. In some embodiments, the term refers to DNA, RNA, or other polynucleotides contained in, for example, tissue or fluid isolated from a patient (including without limitation plasma, serum, cerebrospinal fluid, lymph, tears, saliva and tissue sections), from preserved tissue (such as FFPE sections), or from components of cell culture in vitro, as well as samples from the environment, any plant, animal or viral material. Any sample containing nucleic acids (such as genomic DNA from tissue culture cells or from a tissue sample) may be employed in the present technique.

[0034] The term "nucleic acid sample" as used herein means a sample containing nucleic acids. A nucleic acid sample may be complex in that it contains a plurality of different molecules including sequences. Nucleic acid samples from mammals (e.g., mouse or human) are of a complex nature. A complex sample may have 10 4 、10 5 、10 6 or 10 7 or more different nucleic acid molecules. Also, a complex sample may consist of only 2 or 3 molecules, in which case the molecules together are 10 4 、10 5 、10 6 or 10 7It has more nucleotides than [a certain number]. The term "complexity" generally refers to the total number of different sequences in a population (such as fragments, adapters, or adapter-ligated fragments). For example, if a population has four different sequences, that population has 4 complexities. A population may have at least 4, at least 8, at least 16, at least 100, at least 1,000, at least 10,000, or at least 100,000 or more complexities, depending on the desired outcome.

[0035] The term "nucleotide" refers to naturally occurring nucleotides that include guanine, cytosine, adenine, thymine, and uracil (G, C, A, T, and U, respectively), as well as modified pyrimidine and purine derivatives, and other non-naturally occurring moieties, including not only known purine and pyrimidine bases but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose, or other heterocycles. In addition, the term "nucleotide" may include these moieties containing haptens or fluorescent labels, and may also include other sugars, not just conventional ribose and deoxyribose sugars. Modified nucleotides also include modifications on the sugar moiety, for example, in which one or more hydroxyl groups are substituted with halogen atoms or aliphatic groups, and are functionalized as ethers, amines, etc.

[0036] The terms “nucleic acid” and “polynucleotide” are used interchangeably herein to describe polymers containing nucleotides of any length (composed of nucleotides (e.g., deoxyribonucleotides or ribonucleotides), e.g., more than about 2 bases, more than about 10 bases, more than about 100 bases, more than about 500 bases, more than about 1000 bases, up to about 10,000 bases or more) and may be produced naturally, chemically, enzymatically, or synthetically. The terms include polymers having PNA, LNA, or UNA. While DNA and RNA have deoxyribose and ribose sugar backbones, respectively, the backbone of PNA is formed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA, various purine and pyrimidine bases are linked to the backbone by methylene carbonyl bonds. Locked nucleic acids (LNA), often called inaccessible RNA, are modified RNA nucleotides. The ribose portion of LNA nucleotides is modified by additional crosslinks connecting the 2' oxygen and 4' carbon atoms. These crosslinks "lock" the ribose in the 3'-endo (North) conformation, which is often found in type A double helix. LNA nucleotides can be mixed with DNA or RNA residues in oligonucleotides whenever desired. The term "unstructured nucleic acid," or "UNA," refers to nucleic acids containing non-native nucleotides that bind to each other in a less stable state. For example, unstructured nucleic acids may contain G' and C' residues, which correspond to non-native forms (i.e., analogs) of G and C that base pair with each other in a less stable state, but retain the ability to base pair with naturally occurring C and G residues, respectively.

[0037] The term "base" refers to a naturally occurring, substituted, modified, or altered variant or analogue of a substituted or unsubstituted nitrogen-containing aromatic heterocycle of the type commonly found in nucleic acids, which is capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with appropriately complementary bases.

[0038] The term "linker" refers to one or more divalent groups, such as -C(O)NH-, -C(O)O-, -NH-, -S-, -S(O)n (where n is 0, 1, or 2), -O-, -OP(O)(OH)O-, -OP(O)(O - )O-, alkanediyl, alkendiyl, alkynediyl, arenediyl, heteroarenediyl, and combinations thereof, which function as molecular bridges covalently bonded between two other groups. Linkers may have pendant side chains or pendant functional groups (or both).

[0039] The term "reporter" refers to a chemical component that can directly or indirectly generate a detectable signal. Examples of reporters include fluorescent dye groups, radioactive labels, or groups that emit signals through chemiluminescence or bioluminescence means. Examples of fluorescent dye groups include zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, ALEXA FLUOR 350, ALEXA FLUOR 405, ALEXA FLUOR 430, ALEXA FLUOR 488, ALEXA FLUOR 514, ALEXA FLUOR 532, ALEXA FLUOR 546, ALEXA FLUOR 555, ALEXA FLUOR 568, ALEXA FLUOR 568, ALEXA FLUOR 594, ALEXA FLUOR 610, ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660, ALEXA FLUOR 680, ALEXA FLUOR 700, ALEXA FLUOR This includes 750 and squaline dyes. Additional examples of fluorescent dye reporters that may be used in some embodiments of the present invention are disclosed in Non-Patent Document 1 (2005) and Patent Documents 1 and 2, which are incorporated herein by reference. Examples of radiolabels that may be used as reporters in some embodiments of the present invention are known in the art. 35 S, 3 H, 32 P or 33 Examples include P. Examples of reporters that function by chemiluminescence or bioluminescence and may be used as reporters in some embodiments of the present invention are described in Non-Patent Documents 2 (1989), 3 (1989), 4 (1996), and 5 (1983), which are incorporated herein by reference.

[0040] As used herein, the term “oligonucleotide” refers to a single-stranded multimer of nucleotides, generally ranging in length from about 2 to 200 nucleotides to generally up to 500 nucleotides. Oligonucleotides may be synthetically or enzymatically formed, and in some embodiments, they generally have a length of about 30 to 150 nucleotides. Oligonucleotides may comprise ribonucleotide monomers (i.e., oligoribonucleotides), or deoxyribonucleotide monomers, or both ribonucleotide monomers and deoxyribonucleotide monomers. In some embodiments, the oligonucleotides may have lengths of, for example, 10 to 20, 11 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150, or 150 to 200 nucleotides.

[0041] The term "primer" refers to a natural or synthetic oligonucleotide that can act as a starting point for nucleic acid synthesis when forming a double helix with a polynucleotide template, such as a polynucleotide target, and can be extended along the template from its 3' end so that an extended double helix is ​​formed. The term "extend" as used herein refers to the extension of a primer by the addition of nucleotides using a primer elongating enzyme. When a primer annealed to a nucleic acid is extended, the nucleic acid acts as a template for the extension reaction. The sequence of nucleotides added during the extension process is determined by the sequence of the polynucleotide template. Primers can be extended by primer elongating enzymes such as DNA polymerase and reverse transcriptase. Reverse transcriptase is an RNA-dependent DNA polymerase with a deoxyribonucleotide incorporated on the opposite side of the RNA template. The resulting cDNA (complementary DNA) can then be used by DNA-dependent DNA polymerase to act as a DNA template in subsequent PCR. Primers are generally of a length suitable for use in the synthesis of primer extension products, typically ranging from 8 to 100 nucleotides in length, such as 10-75, 15-60, 15-40, 18-30, 20-40, 21-50, 22-45, and 25-40, and more commonly ranging from 18-40, 20-35, and 21-30 nucleotides, or any length within the ranges described above. Typical primers may also be in the range of 10-50 nucleotides in length, such as 15-45, 18-40, 20-30, and 21-25, or any length within the ranges described above. In some embodiments, primers typically have no more than 10, 12, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, or 70 nucleotides in length.

[0042] Primers are typically single-stranded for amplification, or they may be supplied to the mixture in double-stranded form. In the case of double-stranded primers, they are usually first processed to separate their strands before being used to prepare the extension product. Therefore, the primer is complementary to the template and complexes with the template by hydrogen bonding or hybridization to give a primer / template complex for the initiation of synthesis by polymerase, which is extended during DNA synthesis by the addition of covalently bonded bases linked at the 3' end, complementary to the template. The terms "reverse primer" and "forward primer" refer to primers that hybridize to different strands in a double-stranded DNA molecule, in which case the extension of the primer by polymerase is directed toward the other primer. cDNA synthesis can be primed with reverse transcriptase (RT) primers. For example, an oligonucleotide comprising a series of deoxythymidine nucleotides (oligo(dT)) can anneal to the 3' poly-A tail of an RNA transcript. Alternatively, the RT primer can be annealed to multiple sequence-specific sites within the RNA (target-specific primer).

[0043] A "pair" of primers refers to forward and reverse primers designed to amplify a double-stranded polynucleotide target. In some embodiments, the composition, method, and kit comprises a highly multiplexed set of target-specific primers (e.g., at least 5 pairs of target-specific primers, or at least 10 pairs, or at least 20 pairs, or at least 50 pairs, or at least 100 pairs, or at least 200 pairs, or at least 500 pairs, or at least 1,000 pairs, or at least 2,000 pairs, or at least 5,000 pairs, or at least 10,000 pairs, or at least 20,000 pairs, or more).

[0044] The term "primer extension reagent" refers to any reagent necessary or suitable for performing a primer extension reaction (such as polymerase chain reaction (PCR)) on a polynucleotide molecule, such as a polynucleotide target. Primer extension reagents generally contain nucleotides in a mixture with primers, a heat-stable polymerase or reverse transcriptase, and a suitable buffer. Depending on the enzyme used, ions (e.g., Mg) may be present. 2+ ) may also be present. cDNA synthesis is prepared using reverse primers annealed to the 3' polyA tail of an RNA transcript (oligo(dT)) or to multiple sequence-specific sites within the RNA (randomer, target-specific primer).

[0045] As used herein, the term “universal sequence” refers to a sequence common to two or more nucleic acid molecules in a set or population, preferably substantially all nucleic acid molecules in the set or population, where the nucleic molecules also have distinct parts (such as the target portion in a set of polynucleotide target amplicons). The universal sequence may be present in different elements of the set or population of molecules, thereby enabling common processing of different molecules. Non-limiting examples of universal sequences include sequences identical or complementary to the capture sequence of a flow cell. Similarly, a universal sequence can enable amplification of multiple different nucleic acids using a population of universal amplification primers complementary to a portion of the universal sequence (e.g., a universal primer binding site).

[0046] In some embodiments, the target-specific primers described herein have a 5' region comprising a universal sequence, thereby allowing the universal sequence or its complements to be incorporated into the target amplicon generated in a previous PCR step. Subsequent amplification in a later PCR step using a blocked universal primer, which is unblocked and hybridizes to the universal sequence, can be used to universally amplify the target amplicon present in the PCR mixture.

[0047] The terms “upstream” and “of 5'” are interchangeable when referring to positions within a nucleic acid sequence, referring to relative positions further toward the 5' end of the sequence. The terms “downstream” and “of 3'” are interchangeable when referring to positions within a nucleic acid sequence, referring to relative positions further toward the 3' end of the sequence.

[0048] As used herein, the terms “hybridize” or “hybridization” refer to the process by which a strand of nucleic acid binds to a complementary strand through base pairing. The terms “hybridize” or “hybridization” also encompass the process by which a nucleic acid strand anneals with a second complementary nucleic acid strand under normal hybridization conditions to form a stable double helix (whether homoduplex or heteroduplex) and does not form a stable double helix with unrelated nucleic acid molecules under the same normal hybridization conditions. As used herein, the terms “double-stranded” or “double-stranded” refer to two complementary polynucleotides that have been base-paired (i.e., hybridized) to one another. Double helix formation is achieved in a hybridization reaction by annealing two complementary nucleic acid strands. Hybridization processes can be made highly specific by adjusting the hybridization conditions (often called hybridization stringency) under which the hybridization reaction occurs, resulting in hybridization between two nucleic acid strands not forming a stable double helix unless these two strands contain a specific number of nucleotides in substantially or completely complementary specific sequences. "Normal hybridization" or "normal stringency conditions" can be easily determined for a given hybridization reaction.

[0049] The term "complementary" refers to two nucleic acids that hybridize with each other under highly strict conditions. The term "perfectly complementary" refers to a double-stranded nucleic acid where each base of one nucleic acid base pairs with a complementary nucleotide in the other nucleic acid. Often, two complementary sequences have at least 10 (e.g., at least 12 or 15) complementary nucleotides. In contrast, if two nucleic acids are "incomplementary," they do not hybridize with each other, but some sequence matching (i.e., non-complementary to a degree of less than 100%) may be acceptable, as long as the two strands remain in single-stranded form under the conditions defined above for use in this method.

[0050] The term "amplify" refers to the process of synthesizing a nucleic acid molecule complementary to one or both strands of a template nucleic acid, such as a polynucleotide target. Amplifying a nucleic acid molecule may include denaturing the template nucleic acid, annealing the primers to the template nucleic acid at a temperature below the melting point of the primers, and enzymatically stretching from the primers to produce an amplification product. Each of the denaturation, annealing, and stretching steps can be performed once or more times. In some cases, the denaturation, annealing, and stretching steps are performed multiple times (often rapidly) to increase the amount of amplification product, although rapid amplification is not required in this method. Amplification typically requires deoxyribonucleoside triphosphate, a DNA polymerase enzyme, and appropriate buffers and / or cofactors for optimal activity of the polymerase enzyme. The term "amplification product" or "amplicon" refers to the nucleic acid sequence produced by the amplification process as defined herein. Reverse transcription is a linear amplification reaction that uses a specialized DNA polymerase (reverse transcriptase) to transcribe RNA into cDNA (complementary DNA) using deoxyribonucleoside triphosphate. When RT-PCR is performed in a single vessel, the buffers and cofactors must support the optimal activity of both the reverse transcriptase and the PCR enzyme.

[0051] The term "identifier" refers to a sequence of nucleotides that can be used to a) identify and / or track the origin of polynucleotides in a reaction, b) count how many times the initial molecule has been sequenced, and c) pair sequence reads from different strands of the same molecule.

[0052] The term "adapter" refers to a nucleic acid attached to a polynucleotide, polynucleotide target, or target amplicon in preparation for sequencing. Adapters can be attached by primer extension, ligation, or other techniques. Adapters may be single-stranded or double-stranded and may comprise DNA, RNA, and / or artificial nucleotides. Adapters may be located at the end, middle, or within the polynucleotide. Adapters can have one or more functional regions attached to the polynucleotide, such as providing a primer binding site for a subsequent primer extension step or sequencing, or providing an identifier. For example, an adapter may include a universal priming site containing a universal primer and / or a priming site for sequencing, and / or a capture site for an NGS sequencing system.

[0053] The term "polynucleotide target" refers to the polynucleotide in question. An isolated polynucleotide target molecule refers to a single molecule present in a composition that does not contain other polynucleotide target molecules.

[0054] The term "region" refers to a sequence of nucleotides that may be single-stranded or double-stranded.

[0055] The definitions of other terms may become clear throughout the specification or may be inferred from the specification.

[0056] The precise nucleotide sequences of target-specific primers and universal primers are generally not critical to this technology and may be selected by the user based on the teachings of this disclosure.

[0057] Unless otherwise defined, scientific and technical terms used herein have the same meanings as those generally understood by those skilled in the art of the disclosure.

[0058] All patents and publications referenced herein are expressly incorporated by reference, including all sequences disclosed in such patents and publications.

[0059] In one aspect, the disclosure provides a method for carrying out a multi-step primer extension reaction in a closed container by preparing a primer extension mixture in the container, the mixture comprising i) a polynucleotide target, ii) an unblocked primer, and iii) a blocked primer. The mixture generally comprises other primer extension reagents such as deoxyribonucleotide triphosphate (dNTP), DNA polymerase or other primer extension enzymes, and buffers. In some embodiments, the unblocked or blocked primer comprises a target-specific primer. The target-specific primer comprises a 5' region and a 3' region, the 3' region comprising a target-specific sequence, and the 5' region comprising a universal sequence. The universal primer comprises a universal sequence or a complementary sequence, and has a photocleavable protecting group at its 3' end. In some embodiments, the container is closed after the preparation of the mixture, and the preceding polymerase chain reaction is carried out in the mixture to produce a target amplicon or target cDNA. The blocked primer can be unblocked in the mixture to produce an unblocked primer comprising a universal sequence or a complementary sequence. In some embodiments, deblocking is performed without opening the container. The subsequent primer extension reaction can be carried out with the deblocked primer and target amplicon or target cDNA, in which case the deblocked primer amplifies the target amplicon. In some embodiments, deblocking and / or the subsequent primer extension reaction is carried out by photocleaving the protecting group from the blocked universal primer, such as by exposing the blocked primer to ultraviolet light in a closed container.

[0060] In additional aspects, the technology relates to a novel composition for carrying out a primer extension reaction, the composition comprising a) a polynucleotide target, b) an unblocked primer for the pre-stage, and c) a blocked primer for the post-stage. In some aspects, the target-specific primer comprises a 5' region and a 3' region, the 3' region comprising a target-specific sequence and the 5' region comprising a universal sequence. In other aspects, the universal primer comprises a universal sequence or a portion thereof and a photocleavage-type protecting group. In some aspects, the composition is prepared in a container that is closed after the composition is prepared. In other aspects, the universal primer is unblocked by exposure to ultraviolet light or other photocleavage techniques.

[0061] In other aspects, the technology relates to a kit for carrying out the method, as described above. In some embodiments, the kit may comprise a composition for a multi-step primer extension reaction, as described above. In some embodiments, the kit may comprise a mixture comprising an unblocked primer, such as a target-specific primer or a reverse transcriptase primer, and a blocked primer, such as a universal primer or a target-specific primer. In some embodiments, the kit comprises a container for housing the mixture of the unblocked target-specific primer and the blocked universal primer.

[0062] In some embodiments, the kit comprises a container that holds a mixture of unblocked RT primers and blocked target-specific primers.

[0063] In some embodiments of this method and composition, the preceding primer is present at a concentration in the range of 0.01 to 0.5 μM, and the subsequent primer is present at a concentration in the range of 0.2 to 1 μM. In some embodiments for multiplex PCR, the target-specific primer is present at a concentration in the range of 0.01 to 0.5 μM, and the universal primer is present at a concentration in the range of 0.2 to 1 μM. In some embodiments for RT-PCR, the RT primer is present at a concentration in the range of 0.01 to 0.5 μM, and the blocked target-specific primer is present at a concentration in the range of 0.2 to 1 μM.

[0064] The compositions, methods, and kits can be used to perform multi-step primer extension reactions against polynucleotide targets such as genomic DNA, mitochondrial DNA, messenger RNA, and microRNA. Polynucleotide targets can be obtained from virtually any organism, including but not limited to plants, animals (e.g., reptiles, mammals, insects, worms, fish, etc.), tissue samples, bacteria, fungi (e.g., yeast), phages, viruses, cadaveric tissue, and archaeological / ancient specimens. In some embodiments, the sample may include polynucleotide targets from mammalian cells such as human, mouse, rat, or monkey cells. The sample may be obtained from cultured cells of clinical specimens (e.g., tissue biopsy, scrape, or wash) or cells of forensic specimens (e.g., cells from specimens collected at a crime scene). In some embodiments, the polynucleotide targets may be obtained from biological specimens such as cells, tissues, bodily fluids, and excretions. The body fluids covered include, but are not limited to, blood, serum, plasma, saliva, mucus, sputum, cerebrospinal fluid, pleural fluid, tears, lactal duct fluid, lymph, sputum, synovial fluid, urine, amniotic fluid, and semen. In certain embodiments, the body fluids may be obtained from a subject (e.g., a human).

[0065] In some embodiments, the polynucleotide target comprises DNA or RNA obtained from a clinical sample (e.g., a patient with or suspected of having a disease or condition such as cancer, inflammatory disease, or pregnancy). In some embodiments, the sample may be prepared by extracting the polynucleotide target from a stored patient sample (e.g., a formalin-fixed paraffin-embedded tissue sample). In some embodiments, the patient sample may be a sample of cell-free circulating DNA from a body fluid (e.g., peripheral blood). In some embodiments, the polynucleotide target used in the preceding step of this method is unamplified DNA that has not been denatured beforehand. In other embodiments, the polynucleotide target in the sample may be partially fragmented (e.g., as in the case of FFPE samples and circulating cell-free DNA (cfDNA) (e.g., ctDNA)). In some embodiments, the composition, method, and kit can be used to perform multi-stage RT-PCR on polynucleotide targets from virtually any organism or sample type, from RNA containing poly(A) fragmented mRNA.

[0066] Subsequent primer with blocked 3' end In some embodiments, the subsequent primer is a composition relating to chemical formula I,

[0067] [ka]

[0068] In the formula, R1 is either H or OH.

[0069] The base in chemical formula I is cytosine, uracil, thymine, adenine, or guanine, or modified pyrimidine and purine derivatives thereof. The base may be any of the substituted or unsubstituted nitrogen-containing aromatic heterocycles of the kind commonly found in nucleic acids, and may be a natural, substituted, modified, or altered variant or analog thereof capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with a suitable complementary base.

[0070] The cleavable termination moiety in chemical formula I is a group that confers polymerase termination property to the compound. In some embodiments, the cleavable termination moiety is a group that confers polymerase termination property to the compound.

[0071] [ka]

[0072] The part relating to the formula, where R3 is alkyl (C≦8) or substituted alkyl (C1-8), and R4 is hydrogen, hydroxy, halo, amino, nitro, cyano, azide, or mercapto, and is alkyl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or a substituted form of any of these groups. (version) where R5 and R6 are independent of each other and are hydrogen, hydroxy, halo, amino, nitro, cyano, azide or mercapto, and are alkyl (C≦6), alkenyl (C≦6), alkynyl (C≦6), aryl (C≦6), aralkyl (C≦8), heteroaryl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or substituted forms of any of these groups, and the chemical formula

[0073] [ka]

[0074] or

[0075] [ka]

[0076] The group is such that X is -O-, -S-, or -NH-, or an alkanediyl (C≦12), alkenediyl (C≦12), alkynediyl (C≦12), or a substitution of any of these groups; Y is -O-, -NH-, alkanediyl (C≦12), or a substitution of alkanediyl (C≦12); n is an integer from 0 to 6; m is an integer from 0 to 6, or a linker reporter, or a salt, tautomer, or an optical isomer thereof.

[0077] Any linker in chemical formula I is one or more divalent groups, such as -C(O)NH-, -C(O)O-, -NH-, -S-, -S(O)n (wherein n is 0, 1, or 2), -O-, -OP(O)(OH)O-, -OP(O)(OH) - This refers to those that function as molecular bridges covalently bonded between two other groups, such as O-, alkanediyl, alkendiyl, alkynediyl, arenediyl, heteroarenediyl, and combinations thereof. Some linkers have a pendant side chain or a pendant functional group (or both). Any reporter is a chemical part that can directly or indirectly produce a detectable signal. Examples of reporters include fluorescent dye groups, radiolabels or groups that emit a signal through chemiluminescence or bioluminescence means. In some embodiments, reporters are selected from the group consisting of xanthenes, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliproteins, and their derivatives.

[0078] The primer in chemical formula I is an oligonucleotide capable of forming a double helix with a polynucleotide target. In some embodiments, the primer has a length of 8 to 100 nucleotides, or a length of 10 to 75, 15 to 60, 15 to 40, 18 to 30, 20 to 40, 21 to 50, 22 to 45, or 25 to 40 nucleotides, or any other length within any other range disclosed herein.

[0079] In some embodiments, the universal primer is (a) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyuridine, (b) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyadenosine, (c) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxy The formula comprises a 3' terminal nucleotide selected from the group consisting of ciguanosine, (d) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxycytidine, (e) 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxythymidine, and mixtures thereof, wherein the nucleotide is optionally substituted with a linker and / or reporter. Exemplary mixtures include nucleotide(a) and (b), nucleotide(a), (b) and (c), nucleotide(a), (b), (c) and (d), nucleotide(a), (b), (c), (d) and (e), nucleotide(b) and (c), nucleotide(b), (c) and (d), nucleotide(b), (c), (d) and (e), nucleotide(a) and (c), nucleotide(a) and (d), nucleotide(a) and (e), nucleotide(a), (b) and (d), nucleotide(a), (c ) and (d), nucleotide (a), (c), (d) and (e), nucleotide (a), (b), (d) and (e), nucleotide (a), (b), (c) and (e), nucleotide (b) and (d), nucleotide (c) and (d), nucleotide (b) and (e), nucleotide (c) and (e), nucleotide (b), (c) and (e), nucleotide (b), (d) and (e), nucleotide (c), (d) and (e), nucleotide (d) and (e), and any other mixtures. Methods, compositions, and kits for multiplex and multi-stage PCR In another aspect, this disclosure provides methods and compositions for improving the efficiency of multiplex nucleic acid amplification. This disclosure also relates to reagents and methods for improving the efficiency of multistage nucleic acid amplification (particularly the performance of two or more amplification reactions designed to occur sequentially in the same reaction mixture or vessel). In particular, it provides compositions that have reduced the formation of primer dimers and abnormal amplification products. Blocked primers do not form extendable double strands before UV deblocking. After UV deblocking, they become primer extendable primers. Such primers are particularly useful when the preceding and succeeding amplification reactions occur in a single reaction mixture or vessel. Additional information relating to multiplex and multistage PCR amplification reactions and reagents can be found in Patent Document 3, which is incorporated herein by reference in its entirety.

[0080] In another aspect, this technology relates to multi-stage RT-PCR using an unblocked RT primer as the preceding primer and a blocked primer as the succeeding primer. For example, the succeeding primer may be a target-specific primer having a photocleavage-type protecting group at its 3' end. In some embodiments, a polyribonucleotide target is reverse transcribed in the preceding stage by reverse transcriptase using an unblocked RT primer to generate target cDNA. Examples of RT primers include oligo(dT) primers, randommers (N6-Nn, where n may be an integer such as 7, 8, 9, or 10), or target-specific RT primers. The target cDNA is then amplified during the subsequent PCR using a target-specific primer that has been unblocked by photocleavage of its protecting group.

[0081] This technology is particularly relevant to multiplex nucleic acid amplification, in which two or more target sequences are amplified in parallel. This is typically achieved by including more than one pair of polynucleotide target-specific primers in a single nucleic acid amplification reaction.

[0082] This technology also relates to multi-stage nucleic acid amplification, where two or more separate amplification reactions occur. Typically, the first amplification reaction utilizes a target-specific primer that amplifies a polynucleotide target molecule. The target-specific primer contains a 5' region and a 3' region, with the 3' region containing the target-specific sequence and the 5' region containing the universal sequence. The universal sequence is incorporated into the amplification product as the reaction proceeds. In the subsequent amplification reaction, the amplification product of the previous amplification is amplified using a universal primer that contains a universal sequence or a portion thereof sufficient to hybridize with a complement of the universal sequence.

[0083] This method typically includes multiple primer extension cycles within each step. For example, the pre-stage may include at least 3, 4, 5, 6, 7, 8, 9, 10 or more primer extension cycles and / or up to 20, 18, 16, 14, 12 or fewer PCR cycles. Similarly, the post-stage may include at least 3, 4, 5, 6, 7, 8, 9, 10 or more primer extension cycles and / or up to 20, 18, 16, 14, 12 or fewer primer extension cycles. The method may also include additional primer extension steps before or after the pre-stage and / or post-stage. For example, the pre-stage may be preceded by a primer extension step to supply higher quality input polynucleotides for the pre-stage, and the post-stage may be followed by a PCR step to supply higher quality output polynucleotides for sequencing or other applications.

[0084] In some embodiments, the subsequent amplification includes a universal primer incorporating an additional sequence, which may be required for further downstream processing and identification purposes. Thus, universal amplification is governed by the fact that the subsequent amplification occurs independently of the specific target sequence of the initial target molecule being amplified. Universal amplification relies on the incorporation of an additional sequence (a universal sequence, as described herein) that can act as a primer binding site in the subsequent amplification into the amplification product from the previous amplification reaction. Thus, the primer region of the primer in the subsequent amplification corresponds to the universal sequence. A primer containing such a primer region is referred to herein as a “universal primer”.

[0085] The universal primers of this technology are equipped with a photocleavable protecting group at their 3' end. Blocked universal primers are inactive with respect to PCR amplification, even if present during the PCR amplification step. Because the 3' protecting group of this technology is photocleavable, blocked universal primers can be removed by exposure to ultraviolet light or other photocleavage techniques. Exposure to ultraviolet light removes the protecting group, generating a universal primer that is active with respect to PCR amplification. Therefore, the universal primers of this technology can be blocked and substantially inactive even if present before target-specific PCR amplification, and can then be activated by exposure to ultraviolet light before the subsequent universal PCR amplification step.

[0086] The polynucleotide targets to be amplified by this technology are not generally limited. Any suitable polynucleotide target molecule may be amplified using the reagents and methods of this technology. Multiple different polynucleotide target molecules may be targeted. This may involve the use of multiple polynucleotide target-specific primer pairs. Therefore, the term polynucleotide target generally refers to a desired sequence of nucleic acid molecules to be amplified, whether as part of the initial polynucleotide target molecule present before amplification begins, or as a polynucleotide target amplicon molecule generated during amplification.

[0087] A polynucleotide target is a molecule comprising a DNA molecule or an RNA molecule, or a molecule derived from a DNA molecule or an RNA molecule. The RNA may be obtained from the same sample type as the DNA, as described above. The RNA may be messenger RNA (mRNA), microRNA (miRNA), etc. In some embodiments, the RNA is reverse transcribed using reverse transcriptase to form a complementary DNA (cDNA) molecule that can be amplified using this technique.

[0088] The target-specific primer pairs in this technology are designed to amplify polynucleotide targets and generally incorporate universal sequences. These universal sequences do not hybridize with the initial polynucleotide target molecule. This function is provided by the target-specific 3' region of the target-specific primer. However, if the universal sequences are incorporated into the amplification product, they (or their complements) can then act as primer-binding sites to which the universal primers hybridize in subsequent amplification steps.

[0089] According to some embodiments, a subsequent PCR step for universal amplification may also be used to include one or more adapter sequences in the subsequent amplicon. The adapter sequence may be any sequence suitable for downstream processing, which enables the detection and / or quantification of its polynucleotide target or amplicon from the sample. For example, an adapter sequence complementary to an oligonucleotide immobilized on a suitable solid surface may allow for the incorporation of such an adapter. Other applications rely on adapters that hybridize to oligonucleotides in liquid. Adapters may be useful for sequence-based or sequencing-based analysis. In some embodiments, the adapter sequence may be any adapter sequence suitable for high-volume nucleic acid sequencing. Such sequencing is typically and preferably performed using a next-generation sequencing (NGS) platform.

[0090] In some embodiments, the universal primer further comprises one or more primer binding sites. For example, a first (or forward) universal primer may have a first primer binding site, and a second (or reverse) universal primer may have a second primer binding site, and the first and second primer binding sites are configured to bind to different primers (e.g., the first and second primer binding sites do not have substantially the same sequence and are substantially complementary). The first and / or second primer binding sites may be sequencing primer binding sites, capture primer binding sites, or a combination thereof. For example, a first universal primer may have a first flow cell amplification primer binding site, and a second universal primer may have a second flow cell amplification primer binding site. When the first primer binding site is a sequencing primer binding site, in some embodiments, the first universal primer further comprises an identifier upstream of the first primer binding site. For example, the first universal primer may have a universal capture sequence upstream of the identifier. When the second primer binding site is a sequencing primer binding site, in some embodiments the second universal primer further has an index downstream of the second primer binding site. For example, the second universal primer may have a universal capture sequence or its complement downstream of the identifier. When the first universal primer does not have an index, in some embodiments the universal capture site is located upstream of the first primer binding site.

[0091] In some embodiments, various primers of the Technology may also be used to affix one or more identifiers (also called indices or barcodes) to the amplified product. With respect to some sites of the Technology relating to the target-specific primers used in the previous amplification, sample identifiers and / or molecular identifiers are preferably included in the primers. In certain embodiments, the identifiers may have lengths ranging from 2 to 36 nucleotides, or 6 to 30 nucleotides, or 8 to 20 nucleotides. In some embodiments, the identifiers may include a “degenerate base region” or “DBR,” where “degenerate base region” and “DBR” refer to a type of molecular identifier that has sufficient complexity to help distinguish between fragments to which the DBRs are attached.

[0092] The term "sample identifier" refers to a type of molecular identifier that can be attached to a polynucleotide, where the sequence identifies the origin of the polynucleotide (i.e., the sample from which the polynucleotide is derived). For use, each sample is tagged with a different sample identifier sequence (for example, one sequence is added to each sample, with different samples being added to different sequences), and the tagged samples are stored. After the stored samples are sequenced, the origin of the sequence can be identified using the sample identifier sequence. The term "molecular identifier" refers to a type of identifier that can be attached to a polynucleotide, where the sequence identifies individual polynucleotides or their amplicons.

[0093] In some embodiments, the universal primer comprises a first and a second universal primer, wherein the first universal primer has a 5'-3' order and includes (i) a first adapter sequence, (ii) a molecular identifier, and (iii) a universal primer region identical to at least a portion of the first universal sequence (in the 5'-3' direction), and the second universal primer has a 5'-3' order and includes (i) a second adapter sequence, (ii) a sample identifier, and (iii) a universal primer region identical to at least a portion of the second universal sequence (in the 5'-3' direction).

[0094] In some embodiments, the present invention provides a method for performing multiplex and multi-stage PCR reactions in a single reaction mixture. In some embodiments, all amplification steps (i.e., pre- and post-PCR steps) in the PCR mixture, starting with a polynucleotide target and including the generation of a related amplification product containing a universal sequence, are performed without the need to separate, remove, or add components. In some embodiments, there is no need to perform a target-specific amplification step in a mixture without universal primers, or to add universal primers between the pre- and post-steps, or to purify the previous amplification product before universal amplification. In some embodiments, all the PCR reagents required for the method (i.e., to generate further amplification products) are combined before the preceding amplification steps are performed. Thus, the method can be performed in a single reaction vessel without opening the vessel after all the PCR reaction mixture components have been added. Once the reaction mixture is formed (apart from performing the amplification itself (e.g., thermal cycling)), there is no need to further manipulate or open the reaction vessel until a universal amplification product is generated. Thus, the method can be considered a "closed-vessel" method. This method is highly preferable because it eliminates the need for the user to add a universal primer between the preceding and succeeding steps.

[0095] In some embodiments, all primer extension steps, starting with the polynucleotide target in the mixture and including the generation of the relevant target amplification product containing the universal sequence (i.e., both the preceding and succeeding primer extension steps), are carried out without the need to separate, remove, or add any components. In some embodiments, there is no need to perform a target-specific amplification step in a mixture without universal primers, or to add universal primers between the preceding and succeeding steps, or to purify the previous target amplification product before universal amplification. In some embodiments, all primer extension reagents required for the method (i.e., to generate further amplification products) are combined before the preceding amplification steps are performed. Thus, the method can be carried out in a single reaction vessel without opening the vessel after all the components of the reaction mixture have been added. Once the reaction mixture is formed (apart from the amplification itself (e.g., thermal cycling)), there is no need to further manipulate or open the reaction vessel until the universal amplification product is generated. Thus, the method can be considered a "closed vessel" method. This method is highly preferable because it eliminates the need for the user to add a primer in the subsequent step between the preceding and subsequent steps.

[0096] The method also includes additional steps after the generation of the universal amplification product (i.e., after universal or subsequent amplification). Such methods are not limited to the same reaction mixture or reaction vessel. Such methods may optionally include detection and quantification of polynucleotide target molecules. In some embodiments, the method of the art is used to identify and optionally quantify specific polynucleotide target molecules. In other embodiments, the method further comprises sequencing of further amplification products. Sequencing is usually carried out in parallel, often by using next-generation sequencing (NGS) technology. Sequencing may be carried out in a reaction mixture different from the reaction mixture of the amplification reaction of the art.

[0097] This technology also relates to multi-step RT-PCR reactions in which two or more separate amplification reactions occur in a single vessel. cDNA synthesis is carried out independently of PCR by blocking the 3' end of the target-specific PCR primer with a photocleavage-type protecting group. cDNA synthesis occurs at an optimal constant temperature for the reverse transcriptase, without interference from PCR primers that otherwise nonspecifically interact to produce primer dimers and other artifacts. Therefore, the target-specific PCR primers in this technology may be substantially inactive during cDNA synthesis and then activated by exposure to ultraviolet light before subsequent primer extension reactions (such as PCR). As described above for multi-step mPCR reactions, the RT-PCR method can be carried out in a single vessel without further manipulation.

[0098] This disclosure also provides a kit for performing the method as described herein. In some embodiments, the kit may comprise a composition for multi-step PCR as described above. In some embodiments, the kit may comprise a PCR mixture comprising a target-specific primer and a blocked universal primer having a photocleavable protecting group at its 3' end. The target-specific primer and the blocked universal primer may be in the mixture in a single container. The kit of the technique may further comprise suitable reagents (e.g., buffers) for performing multi-step PCR. The various components of the kit may be in separate containers, or specific suitable components may be pre-combined into a single container as desired. In addition to the reagents described above, the kit may include any of the additional components used in the method described above (e.g., one or more enzymes and / or buffers).

[0099] In some embodiments, the kit may comprise a composition for multi-step RT-PCR as described above. In some embodiments, the kit may comprise an RT-PCR mixture comprising cDNA synthesis primers (oligo(dT) or randommers) and target-specific primers having a photocleavable protecting group at their 3' end. The cDNA synthesis and blocked target-specific primers may be in the mixture in a single container. The kit of this technology may further comprise appropriate reagents for performing multi-step RT-PCR, which may be provided in any of the forms described above.

[0100] In addition to the components described above, the kit may further include instructions for using the kit's components to carry out the method (i.e., instructions for multi-step amplification of polynucleotide targets). Instructions for carrying out the method may be recorded on a suitable recording medium. For example, the instructions may be printed on a substrate such as paper or plastic. Thus, the instructions may be included in the kit as an accompanying document, on the label of the kit container, or on the kit components (i.e., in relation to the packaging or sub-packaging). In other embodiments, the instructions exist as an electronic storage data file on a suitable computer-readable storage medium (e.g., CD-ROM, diskette, etc.). In yet another embodiment, the actual instructions are not present in the kit, and a means for obtaining the instructions from a remote source is provided, for example, via the Internet. An example of this embodiment is a kit that includes a web address from which the instructions can be viewed and / or downloaded. With respect to the instructions, this means for obtaining the instructions is recorded on a suitable substrate.

[0101] Photocleavable protecting group This technology includes primers that are reversibly blocked at their 3' end by a photocleavage protecting group. These blocked primers may be present during the preceding primer extension reaction, but are blocked from extension during this step. After being unblocked, they become extendable during the subsequent primer extension reaction. The photocleavage protecting group in this technology contains nucleotides attached to the 3' end of the subsequent primer, where they block PCR amplification. The blocked primers are substantially inactive to PCR amplification until they are unblocked and activated by exposure to ultraviolet light or other photocleavage techniques. A wide variety of photocleavage protecting groups can be incorporated into the subsequent primer, such as those described in Patent Documents 4, 5, and 6, which are incorporated herein by reference in their entirety. The subsequent primer may have any of these photocleavage protecting groups at its 3' end so that it is substantially inactive to PCR amplification until it is unblocked. In some embodiments, the photocleavage protecting group has a blocking efficiency of about 90% to about 100%.

[0102] Photocleavable protecting groups are designed to reversibly block and terminate DNA synthesis, and then efficiently cleave upon exposure to ultraviolet light, thereby initiating a primer. In some embodiments, the photocleavable protecting group is in the form of a nucleotide compound, including the base adenine, cytosine, guanine, thymine, uracil, or their modified pyrimidine and purine derivatives, such as 7-Hydroxyl-7-deaza-adenine / guanine. In other embodiments, the cleavable group may be derivatized to include a reporter such as a dye. In some embodiments, the base adenine, cytosine, guanine, thymine, uracil, or their modified pyrimidine and purine derivatives may be covalently bonded to a photocleavable protecting group, such as a 2-nitrobenzyl group. In some embodiments, the 2-nitrobenzyl group is derivatized to facilitate the termination of its DNA synthesis. Photocleavage-type protecting groups, such as 2-nitrobenzyl groups, can also be derivatized in some embodiments by covalent bonding to the photocleavage-type protecting group with a fluorescent dye.

[0103] In some embodiments, the photocleavage protecting group comprises a nucleotide base covalently bonded to a 2-nitrobenzyl group, with the alpha-carbon position of the 2-nitrobenzyl group optionally substituted with one alkyl or aryl group. In other embodiments, the 2-nitrobenzyl group may be functionalized to enhance termination and protection properties and the light-catalyzed deprotection rate. In other embodiments, the termination and protection properties of the alpha-carbon substituted 2-nitrobenzyl group, which is attached to the base, occur even if the 3'-OH group on the ribose sugar is unblocked. In some embodiments, the photocleavage protecting group is selected to be well-tolerated by several commercially available DNA polymerases. In some embodiments, the alpha-carbon substituted 2-nitrobenzyl group may also be derivatized to contain a selected fluorescent dye or other reporter.

[0104] Method for preparing photocleavage protecting groups Photocleavage-type protecting groups are forms of nucleotide compounds containing photocleavage-type protective groups designed to rapidly terminate DNA synthesis and cleavage. These are attached to the 3' end of a primer precursor by single-nucleotide elongation of the primer precursor annealed to a template with DNA polymerase, or by template-independent single-nucleotide elongation of the primer precursor with terminal deoxynucleotide transferase (TdT). Therefore, universal primers with photocleavage-type protecting groups are inactive with respect to further elongation.

[0105] In another embodiment, the nucleotide having a photocleavage-type protecting group is a compound with a chemical formula that can be attached to the 3' end of a universal primer,

[0106] [ka]

[0107] In the formula, R1 is H or OH, R2 is H, monophosphate, diphosphate, triphosphate, or thiotriphosphate, the base is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof, the cleavable terminal portion is a group that confers polymerase termination properties to the compound, and any linker is a bifunctional group. The base in chemical formula II is cytosine, uracil, thymine, adenine, or guanine, or a modified pyrimidine or purine derivative thereof. As described above, the base may be any of the substituted or unsubstituted nitrogen-containing aromatic heterocycles of the kind commonly found in nucleic acids, and may be their native, substituted, modified, or altered variants or analogs capable of forming Watson-Crick and / or Hoogsteen hydrogen bonds with a suitable complementary base.

[0108] The cleavage end portion in chemical formula II is a group that confers polymerase termination properties to the compound. Any linker in chemical formula I is one or more divalent groups that function as a molecular bridge covalently bonded between two other groups. Any reporter is a chemical moiety that can directly or indirectly generate a detectable signal. Examples of cleavage end portions, any linkers, and any reporters are given above with respect to chemical formula I, and these exemplary cleavage end portions, any linkers, and any reporters can also be incorporated into chemical formula II.

[0109] Examples Example 1: Production of photocleavage-type 3'-blocked primers In this example, primers were synthesized with a protecting group at their 3' end. Photocleavable blocked primers were generated by single-nucleotide extension of the primer precursor. The primer precursor (number 1-1) was annealed to a DNA template, and a nucleotide containing a photocleavable protecting group (LT-dG) was incorporated into the 3' end of the primer precursor by single-nucleotide extension. The product was purified and analyzed by reverse-phase high-performance liquid chromatography (HPLC). In Figure 2, the leftmost peak shows the products of the annealed primer and template before LT-dG addition, the middle peak shows the single-nucleotide extension product of the primer with added LT-dG, and the rightmost peak shows excess, unincorporated LT-dG.

[0110] Example 2: A photocleavage-type blocked primer can be unblocked by ultraviolet light. In this example, the ability to unblock primers having a protecting group at their 3' end was evaluated. Figure 4 shows HPLC traces of an HPLC universal purification primer with a photocleavable protecting group at its 3' end (main peak on the right) and the universal primer after 10 seconds of exposure to 365 nm UV light (peak on the left). The increase in HPLC mobility is due to the cleavage of the photocleavable protecting group from the 3' end of the primer by ultraviolet light. This demonstrates that photocleavable blocked primers of this technology are capable of being efficiently unblocked by ultraviolet light and subsequently extended by DNA polymerase.

[0111] Example 3: Photocleavage-type blocked primers can only be extended in PCR after exposure to ultraviolet light. In this example, the use of blocked primers for PCR amplification was evaluated. Figure 5 shows Bioanalyzer 2100 images of three PCR products. The lane labeled "PCR with unblocked primers" is for the positive control and shows the amplified product of a 305 bp fragment of gDNA with unblocked primers (number 1-FP and number 1-RP). The lane labeled "PCR with blocked primers" shows the product of an unblocked reverse primer (number 1-RP) and a photocleavage-type blocked forward primer (number 1-F * This is for PCR attempted with ). This lane shows minimal PCR amplification due to blocked primers that cannot become extended PCR amplification. The lane labeled "PCR with UV-exposed blocked primers" shows forward primers (numbers 1-F). * This is for PCR with unblocked primers and photocleavable blocked forward primers after the protecting group on the primer is cleaved by exposure to ultraviolet light. This lane shows amplification of the PCR product after the forward primer has been unblocked and made extendable during PCR amplification. These results demonstrate that the blocked primers of this technique, although not extendable during PCR amplification, can be unblocked by exposure to ultraviolet light and activated to result in extended PCR amplification.

[0112] Example 4: Photocleavage-type blocked primers can only be extended in RT-PCR after exposure to ultraviolet light. In this example, the use of blocked target-specific primers was evaluated in RT-PCR as another embodiment of a multi-step primer extension reaction. Figure 6 shows photocleavage-type blocked β-actin reverse primers (Panel A, R * ) or photocleavage-type blocked No. 1 forward primer (Panel B, F *The image shows a Bioanalyzer 2100 image of the product of a single-container RT-PCR reaction performed in ). The closed tube was exposed to UV for 3 minutes between cDNA synthesis and thermal cycling. Without UV exposure, no target-specific product was generated in any assay, and β-actin R * and number 1 F * However, it is suggested that it remains inactive during both cDNA synthesis and the PCR step. With further control, β-actin R is shown to remain inactive during the period between UV exposure and the first PCR denaturation step (not shown). * It was shown that reverse transcription was prepared (primed). By performing UV exposure at high temperature, nonspecific interactions could be prevented during this short period. The results indicate that the blocked target-specific primers were extended only in RT-PCR after exposure to ultraviolet light.

[0113] Exemplary Embodiments Exemplary embodiments presented in accordance with the subject matter disclosed herein include, but are not limited to, the following:

[0114] Embodiment 1. A method for performing a multi-step primer extension reaction in a closed container. The method comprises a) a step of preparing a primer extension mixture in a container, the mixture comprising i) a polynucleotide target, ii) a primer-extendable pre-primer, iii) a post-primer having a photocleavable protecting group at its 3' end, iV) a primer extension enzyme, and v) a primer extension reagent. The container is closed after the preparation of the mixture. The method also comprises b) a step of performing a pre-primer extension reaction with the pre-primer to generate a target amplicon or target cDNA. The method comprises c) a step of unblocking the post-primer to generate an unblocked post-primer, the unblocking step of which is performed without opening the container. The method also comprises d) a step of performing a post-primer extension reaction with the unblocked post-primer and the target amplicon or target cDNA. The unblocked post-primer is extended by hybridizing with the target amplicon or target cDNA.

[0115] Embodiment 2. The method according to Embodiment 1, wherein the preceding primer comprises a target-specific primer having a 5' region and a 3' region, the 3' region having a target-specific sequence and the 5' region having a universal sequence.

[0116] Embodiment 3. The method according to Embodiment 2, wherein the subsequent primer comprises a universal primer having the universal array or a part thereof.

[0117] Embodiment 4. The method according to Embodiments 1 to 3, wherein the preceding primer comprises a reverse transcriptase (RT) primer.

[0118] Embodiment 5. The method according to Embodiment 4, wherein the subsequent primer comprises a target-specific primer.

[0119] Embodiment 6. The method according to any one of Embodiments 1 to 5, wherein the unblocking step (c) comprises the step of exposing the subsequent primer to ultraviolet light in the closed container.

[0120] Embodiment 7. The blocked primer is a compound relating to chemical formula I,

[0121] [ka]

[0122] The method according to any one of Embodiments 1 to 6, wherein R1 is H or OH, the base is cytosine, uracil, thymine, adenine or guanine, or modified pyrimidines and purine derivatives thereof, the cleavable terminal portion is a group that confers polymerase termination properties to the compound, the optional linker is a divalent group, the optional reporter is a chemical portion that can directly or indirectly generate a detectable signal, and the primer is an oligonucleotide that can form a double helix with a polynucleotide target.

[0123] Embodiment 8. The cleavage-type end portion is a portion relating to the following chemical formula,

[0124] [ka]

[0125] During the ceremony, R3 is an alkyl (C≦8) or substituted alkyl (C1-8). R4 is hydrogen, hydroxy, halo, amino, nitro, cyano, azide, or mercapto, and is alkyl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or a substitution of any of these groups. R5 and R6 are independent of each other and are hydrogen, hydroxy, halo, amino, nitro, cyano, azide, or mercapto, and are alkyl (C≦6), alkenyl (C≦6), alkynyl (C≦6), aryl (C≦6), aralkyl (C≦8), heteroaryl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or substituted forms of any of these groups, and their chemical formula

[0126] [ka]

[0127] or

[0128] [ka]

[0129] It is the basis of, X is -O-, -S-, or -NH-, or an alkanediyl (C≦12), alkenediyl (C≦12), alkynediyl (C≦12), or a substitution of any of these groups. Y is -O-, -NH-, alkanediyl (C≦12), or substitution alkanediyl (C≦12), and n is an integer from 0 to 6. m is an integer from 0 to 6, or a linker reporter. The method according to Embodiment 7, which is a salt, a tautomer, or an optical isomer thereof.

[0130] Embodiment 9. The method according to Embodiment 7, wherein the cleavage-type terminal portion is provided with a 2-nitrobenzyl substituent.

[0131] Embodiment 10. The method according to any one of Embodiments 7 to 9, wherein the primer is selected from oligonucleotides having a length between 8 and 100 nucleotides.

[0132] Embodiment 11. The method according to any one of Embodiments 7 to 10, wherein the base is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, modified pyrimidines and purine derivatives thereof, and mixtures thereof.

[0133] Embodiment 12. A composition for performing a multi-step primer extension reaction, comprising a) a polynucleotide target, b) a primer extensionable pre-primer, and c) a post-primer having a photocleavage-type protecting group at its 3' end, wherein the composition is contained in a container that is closed during preparation.

[0134] Embodiment 13. The composition according to claim 12, wherein the subsequent primer is configured to be unblocked by exposure to ultraviolet light.

[0135] Embodiment 14. The composition according to Embodiment 12 or 13, wherein the photocleavage-type protecting group has a blocking efficiency of about 90% to about 100%.

[0136] Embodiment 15. The composition according to any one of Embodiments 12 to 14, comprising at least 5 pairs of target-specific primers, or at least 5 pairs of target-specific primers, or at least 10 pairs, or at least 20 pairs, or at least 50 pairs, or at least 100 pairs, or at least 200 pairs, or at least 500 pairs, or at least 1,000 pairs, or at least 2,000 pairs, or at least 5,000 pairs, or at least 10,000 pairs, or at least 20,000 pairs.

[0137] Embodiment 16. The composition according to any one of Embodiments 12 to 15, wherein the preceding primer is present at a concentration of 0.01 to 0.5 μM, and the subsequent primer is present at a concentration of 0.2 to 1 μM.

[0138] Embodiment 17. The subsequent primer is 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyuridine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyadenosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyguanosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxycytidine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxythymidine, The composition according to any one of embodiments 12 to 16, further comprising a 3' terminal nucleotide selected from the group consisting of any two, three, four, or five of the above nucleotides, and a mixture thereof.

[0139] Embodiment 18. A method for preparing a photocleavage-type blocked primer, comprising: a) supplying a primer precursor having a 3' end; b)i) forming a double strand of the primer precursor hybridized to a template, wherein the template has a 5' overhang to at least one nucleotide at the 3' end of the primer precursor; and extending the primer precursor at its 3' end by incorporating a nucleotide having a photocleavage-type protecting group having a DNA polymerase; or ii) extending the primer precursor at its 3' end by incorporating a nucleotide having a photocleavage-type protecting group having a template-independent DNA polymerase.

[0140] Embodiment 19. The nucleotide having a photocleavage protecting group is a compound according to chemical formula II,

[0141] [ka]

[0142] During the ceremony, R1 is either H or OH. R2 is H, monophosphate, diphosphate, triphosphate, or α-thiotriphosphate. The bases are cytosine, uracil, thymine, adenine, or guanine, or modified pyrimidines and purine derivatives thereof. The cleavage-type terminal portion is a group that imparts polymerase termination characteristics to the compound. Any linker is a divalent base, The method according to claim 18, wherein any reporter is a chemical portion capable of directly or indirectly generating a detectable signal.

Claims

1. A method for performing a multi-step primer extension reaction in a closed container, a) A step of preparing a primer extension mixture in a container, wherein the mixture is i) Polynucleotide targets, ii) A primer set with a primer extension capability in the preceding stage, iii) A subsequent primer set having a photocleavage-type protecting group at its 3' end, iV) Primer elongation enzyme, and, v) Primer extension reagent The container is provided with the step of being closed after the preparation of the mixture, b) A step of generating a target amplicon or target cDNA by performing a primer extension reaction with the primer set in the preceding step, wherein the primer set in the subsequent step is blocked from extending in the container during the extension reaction of the primer set in the preceding step, c) After the extension reaction of the preceding primer set is completed, the step of unblocking the subsequent primer set to generate an unblocked subsequent primer set, wherein the unblocking step is performed without opening the container. d) The process comprises the step of performing a subsequent primer extension reaction with the primer set after the deblocking and the target amplicon or target cDNA, A method wherein the primer set following the deblocking is hybridized with the target amplicon or target cDNA and extended.

2. The method according to claim 1, wherein the primer set in the preceding step comprises a target-specific primer having a 5' region and a 3' region, the 3' region comprising a target-specific sequence and the 5' region comprising a universal sequence.

3. The method according to claim 2, wherein the primer set in the latter stage comprises a universal primer having the universal array or a part thereof.

4. The method according to claim 1, wherein the primer set in the preceding step comprises a reverse transcriptase (RT) primer.

5. The method according to claim 4, wherein the primer set in the subsequent stage comprises target-specific primers.

6. The method according to claim 1, wherein the deblocking step (c) comprises the step of exposing the subsequent primer set to ultraviolet light in the closed container.

7. The blocked primer is a compound relating to chemical formula I, Chemical formula I 【Chemistry 11】 During the ceremony, R1 is either H or OH. The bases are cytosine, uracil, thymine, adenine, or guanine, or modified pyrimidines and purine derivatives thereof. The cleavage-type terminal portion is a group that imparts polymerase termination characteristics to the compound. Any linker is a divalent base, Any reporter is a chemical component that can directly or indirectly generate a detectable signal. The method according to claim 1, wherein the primer is an oligonucleotide capable of forming a double helix with a polynucleotide target.

8. The aforementioned cleavage-type terminal portion is the part relating to the following chemical formula, 【Chemistry 12】 During the ceremony, R3 is an alkyl (C ≤ 8) or substituted alkyl (C 1-8). R4 is hydrogen, hydroxy, halo, amino, nitro, cyano, azide, or mercapto, and is alkyl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or a substitution of any of these groups. R5 and R6 are independent of each other and are hydrogen, hydroxy, halo, amino, nitro, cyano, azide, or mercapto, and are alkyl (C≦6), alkenyl (C≦6), alkynyl (C≦6), aryl (C≦6), aralkyl (C≦8), heteroaryl (C≦6), acyl (C≦6), alkoxy (C≦6), acyloxy (C≦6), alkylamino (C≦6), dialkylamino (C≦6), amide (C≦6), or substituted forms of any of these groups, and their chemical formula 【Chemistry 13】 or 【Chemistry 14】 It is the basis of, X is -O-, -S-, or -NH-, or an alkanediyl (C≦12), alkenediyl (C≦12), alkynediyl (C≦12), or a substitution of any of these groups. Y is -O-, -NH-, alkanediyl (C≦12), or substitution alkanediyl (C≦12), and n is an integer from 0 to 6. m is an integer from 0 to 6, or a linker reporter. The method according to claim 7, wherein the salt is a salt, a tautomer, or an optical isomer thereof.

9. The method according to claim 7, wherein the cleavage-type terminal portion comprises a 2-nitrobenzyl substituent.

10. The method according to claim 7, wherein the base is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, modified pyrimidines and purine derivatives thereof, and mixtures thereof.

11. The method according to any one of claims 1 to 10, wherein the photocleavage-type protecting group has a blocking efficiency of 90% to 100%.

12. The method according to claim 1, comprising at least five pairs of target-specific primers, or at least 10 pairs, or at least 20 pairs, or at least 50 pairs, or at least 100 pairs, or at least 200 pairs, or at least 500 pairs, or at least 1,000 pairs, or at least 2,000 pairs, or at least 5,000 pairs, or at least 10,000 pairs, or at least 20,000 pairs of target-specific primers.

13. The method according to claim 1, wherein the preceding primer set is present at a concentration of 0.01 to 0.5 μM, and the subsequent primer set is present at a concentration of 0.2 to 1 μM.

14. The aforementioned primer set in the latter stage is 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyuridine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyadenosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxyguanosine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxycytidine, 5-[(S)-1-(5-methoxy-2-nitrophenyl)-2,2-dimethylpropyloxy]methyl-2'-deoxythymidine, The method according to claim 13, further comprising a 3' terminal nucleotide selected from the group consisting of mixtures thereof.

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