Chimeric primers and related methods

Abstract

The present disclosure provides chimeric primers suitable for use in amplifying nucleic acid sequences. In some embodiments, these chimeric primers reduce the formation of primer-dimers and / or off-target amplification products compared to amplification reactions performed using unmodified primers.

Description

[Technical Field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Patent Application Nos. 62 / 955,253 and 62 / 955,260, filed December 30, 2019, the entire contents of which are expressly incorporated herein by reference.

[0002] The present disclosure relates generally to compositions, methods, and kits for reducing non-specific nucleic acid amplification, used in the field of nucleic acid amplification and detection. [Background technology]

[0003] Polymerase chain reaction (PCR) methods are central to various diagnostic methods, e.g., high-throughput SNP genotyping, and serve as the basis for applications in forensic analysis including person identification and paternity testing, infectious disease diagnosis, disease diagnosis and prognosis by NGS, and pharmacogenomic studies aimed at understanding the association between an individual's genetic traits and drug response and disease susceptibility.

[0004] Many PCR methods, particularly multiplex PCR, which amplifies several different products in a single reaction, often suffer from low efficiency because primers can hybridize (or "anneal") to each other rather than to the template to be amplified. These off-target interactions can result in nonspecific amplification products, which are template-independent artifacts of the PCR amplification reaction. Thus, the specificity of primer-based amplification methods depends largely on the specificity of primer hybridization and extension. At the elevated temperatures used in typical PCR amplification, each pair of primers will typically hybridize only to its intended target sequence. However, in multiplex PCR, the large number of different primers and their availability in the reaction medium result in numerous short-lived interactions between primers, which inadvertently trigger primer extension by DNA polymerase. Once formed, these extension products (i.e., primer dimers) are preferentially amplified due to their smaller size.

[0005] Nonspecific primer extension products can compete with the amplification of the desired target sequence, significantly reducing the efficiency of amplification of the desired sequence. One common type of nonspecific amplification product is a template-independent artifact of the amplification reaction, often referred to as "primer dimers." Primer dimers are double-stranded fragments formed when a pair of primers hybridize and extend in a PCR reaction mixture. The resulting extension products serve as templates and are efficiently amplified due to their short length. Therefore, researchers involved in large-scale multiplex PCR assays often must devote significant time and resources to designing, testing, and refining PCR primers and assay conditions. Unfortunately, current methods lack a universal solution (e.g., primer design rules that can be consistently applied to PCR assays to mitigate this problem), requiring considerable trial and error before appropriate assay parameters can be determined. Summary of the Invention

[0006] In a general aspect, the present disclosure provides methods, compositions, and kits that can be used to amplify DNA in DNA-dependent polymerase amplification methods (e.g., using multiplex PCR) while advantageously reducing or eliminating the formation of primer-dimers and / or nonspecific amplification products. While the present disclosure typically refers to PCR, as an example, it is understood that the chimeric primers described herein (and methods for designing such primers) can also be used in exponential rolling circle amplification (ERCA), rolling circle amplification (RCA), multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), real-time quantitative PCR (qPCR), self-sustained sequence replication (3SR), Qβ replicase amplification, and cycle sequencing.

[0007] In one aspect, the present disclosure relates to a method for generating chimeric primers capable of amplifying at least a portion of a template DNA molecule while minimizing or eliminating non-specific amplification, the method comprising the steps of: a) identifying non-specific amplified fragments generated during a PCR assay; b) identifying one or more DNA primers that generate the non-specific amplified fragments; and c) selecting a sequence for at least one chimeric primer; and optionally d) generating the at least one chimeric primer, wherein the chimeric primer is an oligonucleotide comprising DNA and RNA bases and has a sequence identical to that of one of the identified DNA primers except for a first segment in which at least two adjacent DNA bases are replaced by corresponding RNA bases. The chimeric primer may be generated using any oligonucleotide synthesis method known in the art.

[0008] In some embodiments, the first segment is located outside the overlap region that occurs when one or more DNA primers identified in step b) hybridize with one or more other DNA primers to form primer-dimers during a PCR assay. In some embodiments, the overlap region is determined based on hybridization conditions that are compatible with the PCR assay.

[0009] In some embodiments, the corresponding RNA base is located within 15 bases of the 3' end of the sequence, and the sequence has a DNA base at its 3' end.

[0010] In some embodiments, the sequence of the chimeric primer further comprises a second segment comprising at least two adjacent DNA bases replaced by corresponding RNA bases, and the first and second segments are separated by at least two DNA bases. The first and second segments may be located within 15 bases of the 3' end of the sequence of the selected sequence.

[0011] In some embodiments, the sequence of at least one chimeric primer further comprises a third segment comprising at least two adjacent RNA bases replacing corresponding DNA bases in the identified DNA primer, the second and third segments being separated by at least two DNA bases.

[0012] In some embodiments, selecting a sequence for at least one chimeric primer comprises selecting a sequence for a first chimeric primer configured to hybridize to an upstream portion of the template DNA molecule and selecting a sequence for a second chimeric primer configured to hybridize to a downstream portion of the template DNA molecule, wherein the first and second chimeric primers are capable of amplifying at least a portion of the template DNA molecule by PCR.

[0013] In another general aspect, the disclosure provides a method for generating chimeric primers capable of amplifying DNA while minimizing or eliminating the formation of primer-dimers, the method comprising the steps of: a) identifying primer-dimers generated during a PCR assay; b) identifying one or more DNA primers that give rise to the primer-dimers; c) identifying an overlap region generated when the one or more DNA primers hybridize; and d) selecting a sequence for at least one chimeric primer, wherein the chimeric primer is an oligonucleotide comprising DNA and RNA bases and has a sequence identical to that of one of the identified DNA primers except for a first segment in which at least two adjacent DNA bases are replaced by corresponding RNA bases.

[0014] In some embodiments, the first segment is located outside the overlap region that occurs when one or more DNA primers identified in step b) hybridize with one or more other DNA primers during a PCR assay to form primer-dimers. In some embodiments, the overlap region is determined based on annealing conditions (e.g., high or low stringency conditions) that are compatible with the PCR assay.

[0015] In some embodiments, at least one chimeric primer is configured to amplify at least a portion of the template DNA while reducing or eliminating the formation of non-specific amplification products.

[0016] In some embodiments, the PCR assay is a multiplex PCR assay that produces a final product containing a) less than 5% primer-dimer amplification products, b) less than 7% primer-dimer amplification products, or c) less than 10% primer-dimer amplification products.

[0017] In another general aspect, the disclosure provides chimeric primers generated using any of the methods described herein, as well as kits containing same.

[0018] In yet another general aspect, the disclosure relates to a method for amplifying DNA, the method comprising: a) performing a PCR assay using a reaction mixture comprising one or more chimeric primers, a DNA-dependent polymerase, and a template DNA molecule; and b) amplifying at least a portion of the template DNA molecule using the one or more chimeric primers, wherein each of the one or more chimeric primers is an oligonucleotide comprising DNA and RNA bases, and has a sequence including a first segment comprising at least two adjacent RNA bases.

[0019] In some embodiments, at least one of the one or more chimeric primers comprises a sequence having a pair of adjacent RNA bases spanning positions 3 and 4, 7 and 8, or 14 and 15, measured from the 3' end of the chimeric primer, with the remainder of the sequence consisting of DNA bases.

[0020] In some embodiments, the first segment is located within 15 bases of the 3' end of the sequence, and the sequence has DNA bases at its 3' end.

[0021] In some embodiments, the sequence of at least one of the chimeric primers further comprises a second segment comprising at least two adjacent RNA bases, and the first and second segments are separated by at least two DNA bases (e.g., the first segment may span positions 3 and 4, measured from the 3' end of the chimeric primer, and the second segment may span either positions 7 and 8 or 14 and 15).

[0022] In some embodiments, the sequence of at least one of the chimeric primers further comprises a third segment comprising at least two adjacent RNA bases, and the second and third segments are separated by at least two DNA bases (e.g., the first segment may span positions 3 and 4, the second segment may span positions 7 and 8, and the third segment may span positions 14 and 15, measured from the 3' end of the chimeric primer).

[0023] In some embodiments, the reaction mixture comprises two chimeric primers, each chimeric primer being an oligonucleotide comprising DNA and RNA bases, having a sequence comprising a first segment comprising at least two adjacent RNA bases.

[0024] In some embodiments, the reaction mixture comprises a plurality of chimeric primers, wherein the first segment of each chimeric primer is located within 15 bases of the 3' end of the sequence of each chimeric primer, and each sequence has a DNA base at its 3' end. In some embodiments, the sequence of each chimeric primer further comprises a second segment comprising at least two adjacent RNA bases, and the first segment and the second segment are separated by at least two DNA bases. In some embodiments, the sequence of each chimeric primer further comprises a third segment comprising at least two adjacent RNA bases, and the second segment and the third segment are separated by at least two DNA bases. In some embodiments, at least one of the first segment, the second segment, and the third segment is located within 15 bases of the 3' end of the sequence of each chimeric primer.

[0025] In a further embodiment, the one or more chimeric primers in the reaction mixture comprise a forward primer configured to hybridize to an upstream portion of the template DNA molecule and a reverse primer configured to hybridize to a downstream portion of the template DNA molecule, wherein the forward primer and the reverse primer are chimeric oligonucleotides and each comprise a sequence comprising DNA and RNA bases and a first segment comprising at least two adjacent RNA bases.

[0026] In some embodiments, the forward primer and the reverse primer each comprise a sequence having DNA bases at its 3' end, with the first segment of each sequence being located within 15 bases of the respective 3' ends.

[0027] In some embodiments, at least one of the forward primer and the reverse primer further comprises a second segment comprising at least two adjacent RNA bases, and the first segment and the second segment are separated by at least two DNA bases.

[0028] In some embodiments, both the forward primer and the reverse primer each further comprise a second segment comprising at least two adjacent RNA bases, and the first and second segments are separated by at least two DNA bases.

[0029] In some embodiments, the one or more chimeric primers are configured to amplify at least a portion of the template DNA while reducing or eliminating the formation of non-specific amplification products.

[0030] In some embodiments, the one or more chimeric primers are configured to amplify at least a portion of the template DNA while reducing or eliminating primer-dimers and / or off-target amplification.

[0031] In some embodiments, the PCR assay is a multiplex PCR assay that produces a final product that contains less than 5, 6, 7, 8, 9, or 10% primer-dimer amplification products.

[0032] In some embodiments, the PCR assay is a multiplex PCR assay that produces a final product containing a) less than 5% primer-dimer amplification products, b) less than 7% primer-dimer amplification products, or c) less than 10% primer-dimer amplification products.

[0033] In another general aspect, the disclosure provides methods for designing a chimeric primer, the method comprising: a) selecting a DNA primer from a pair of DNA primers configured to amplify at least a portion of a template DNA molecule in a PCR assay; b) selecting a sequence for the chimeric primer, the chimeric primer being an oligonucleotide comprising DNA and RNA bases, the chimeric primer having a sequence comprising a first segment consisting of two adjacent RNA bases, the sequence being identical to the sequence of the DNA primer selected in step a) except for the first segment; and c) optionally generating the chimeric primer (e.g., using any known oligonucleotide synthesis method). In some aspects, the first segment spans positions 3 and 4, 5 and 6, 6 and 7, 7 and 8, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 12 and 13, 12 and 14, or 14 and 15, measured from the 3' end of the chimeric primer. In other embodiments, the first segment may span any two adjacent bases of the chimeric primer.

[0034] In another general aspect, the disclosure provides chimeric primers generated using any of the methods described herein, as well as kits containing same.

[0035] Other embodiments will be readily apparent to those of skill in the art in view of this entire disclosure. [Brief explanation of the drawings]

[0036] [Figure 1] 1 is a table summarizing the results of a series of PCR assays performed using DNA primers or at least one chimeric primer. Shaded boxes indicate primer pairs that resulted in primer dimer formation, where at least one primer in the pair was a chimeric primer. This figure also illustrates the difference in the level of primer dimer formation between chimeric primers with at least two non-adjacent RNA bases (i.e., the "Peleg" approach, described in more detail below) and chimeric primers with at least two adjacent RNA bases according to the present disclosure. [Figure 2] 1 is a table summarizing the results of a series of PCR assays performed using DNA primers (unshaded boxes) or at least one chimeric primer (shaded boxes) with one, two, or three RNA bases 5′ of the overlap region of each primer pair. [Figure 3] 1 is a table summarizing the results of a series of PCR assays performed using standard DNA primers (unshaded boxes) or at least one chimeric primer (shaded boxes), illustrating the relative coverage of the various amplicons generated using these primer pairs. [Figure 4] Three chromatograms analyzing the results of PCR using either a standard universal primer (top) or two different pairs of chimeric primers (middle and bottom) on genomic DNA. As shown in this figure, no nonspecific fragments were detected when the chimeric primer pair was used, indicating increased specificity. [Figure 5]This figure shows three chromatograms analyzing the results of 30 cycles of PCR performed without genomic DNA in the reaction mixture using either a standard universal primer (top row) or two different pairs of chimeric primers (middle and bottom rows). As shown in this figure, no nonspecific fragments were detected when the chimeric primer pair was used. In contrast, the standard universal primers interacted with each other (e.g., formed primer dimers), resulting in nonspecific amplification products. [Figure 6] 1 shows an exemplary workflow for generating chimeric primers according to the present disclosure, highlighting the location of the overlap region in this representative primer pair. [Figure 7] 1 is a table showing an exemplary set of three DNA primers that give rise to two possible primer-dimers in a PCR assay, each with a different overlap region. [Figure 8] 1 is a table summarizing the results of a series of PCR assays performed using pairs of primers in their original form ("All DNA") or in forms modified to incorporate at least one RNA base at a specific position (rows "RNA Test 1" to "RNA Test 5"). [Figure 9] This table summarizes the results of a series of PCR assays performed using multiple pairs of primers, either in their original form ("All DNA") or modified to incorporate RNA base pairs at specific positions (columns "Version 1" to "Version 3"). Shaded boxes indicate examples where DNA primers were replaced by chimeric primers. [Figure 10] 4A-4C are three graphs illustrating the relative amplicon coverage of the chimeric primers shown in FIG. 3. [Figure 11] 5A-5C are three graphs illustrating, respectively, an expanded portion of the graph shown in FIG. 4, showing details of the lower left quadrant. [Figure 12]1 is a table summarizing the relative amplicon coverage observed in PCR assays using chimeric primers at different concentration levels ("MP4": original concentration, and "MP5": optimized concentration). [Figure 13] 1 is a table analyzing primer dimer formation observed in the MP4 study group. [Figure 14] 1 is a table analyzing primer dimer formation observed in the MP5 test group. [Figure 15] FIG. 1 shows three chromatograms analyzing the amplification results when the CFTR chimera and tetra-chimera assays are combined in one multiplex PCR assay (top), and when the CFTR chimera (middle) and tetra-chimera (bottom) assays are run as separate reactions. [Figure 16] Table summarizing primer dimer results when CFTR chimera and tetrachimera assays are combined into one multiplex PCR assay (top row), and when CFTR chimera (middle row) and tetrachimera (bottom row) assays are run as separate reactions. [Figure 17] 1 is a graph showing coverage of the tetra-chimera assay compared with the CFTR chimera assay, both separately and in combination. [Figure 18] 1 is a graph showing coverage of CFTR chimera assays compared with tetra-chimera assays, both separately and in combination. DETAILED DESCRIPTION OF THE INVENTION

[0037] "Polymerase chain reaction" or "PCR" is an enzymatic reaction that amplifies a specific template DNA using one or more pairs of sequence-specific primers for one target. "Multiplex polymerase chain reaction" or "multiplex PCR" is an enzymatic reaction that uses two or more primer pairs for different target templates. If target templates are present in the reaction, the multiplex polymerase chain reaction results in two or more amplified DNA products that are simultaneously amplified in one reaction using a corresponding number of sequence-specific primer pairs.

[0038] Primer dimer formation is a consideration when performing PCR amplification because these off-target amplification products divert system resources (e.g., primers, polymerase, and dNTPs) from the target PCR reaction. This issue is particularly concerning in multiplex PCR, where several primers are present; even a small number of different primers significantly increases the likelihood of unintended cross-reaction and dimer formation. This issue is a major obstacle when building large-scale multiplex PCR assays and also hinders the shortening of turnaround times for smaller multiplex PCR assays. Reducing primer dimer formation therefore requires a substantial investment of resources, time, and effort and must be performed for each assay, resulting in a long time to market. Multiplex PCR assays that produce amplification products containing, for example, >5% primer dimers can increase sequencing costs for customers and lead to suboptimal results. Thus, the development of commercially viable multiplex PCR assays often requires multiple rounds of optimization and iterative primer design to reduce primer-dimer formation to more acceptable levels (i.e., <5%).

[0039] Numerous methods have been proposed to avoid nonspecific amplification, which is often observed in multiplex PCR. For example, U.S. Patent No. 8,460,874 ("Peleg") discloses that ribonucleotides can be incorporated into standard DNA primers used in multiplex PCR to improve specificity. However, while Peleg suggests that adding RNA bases to DNA primers reduces nonspecific amplification, he also explicitly teaches that adjacent ribonucleotides cannot be used to prevent nonspecific amplification in general, and primer-dimer formation in particular. See, for example, Peleg at 3:23-28 (where he cautions that "the incorporation of very few ribonucleotides into DNA primers...can have a beneficial effect on reducing the generation of undesired artifacts arising from in vitro DNA-dependent DNA polymerase amplification reactions, provided the ribonucleotides are not adjacent"). Furthermore, Peleg's approach fails to provide rules that can be consistently applied based solely on the original sequences of a given pair or set of DNA primers (e.g., multiple solutions may be possible, requiring more experimental work than necessary to establish useful parameters). Other known techniques for addressing nonspecific amplification or primer-dimer formation in multiplex PCR have been based on repeated primer redesign or workflows requiring enzymatic steps to remove formed primer-dimers or activate blocked primers.

[0040] Given these and other shortcomings of current methods, new methods for primer design are needed. In particular, methods are needed that can be used to more efficiently design standard and multiplex PCR assays that amplify one or more target DNA sequences in a single reaction mixture while reducing the formation of nonspecific amplification products such as primer dimers. Such methods, as described herein, rely on the use of at least one "chimeric primer" that includes one or more DNA bases and at least one segment containing two or more adjacent RNA bases. In some embodiments, these chimeric primers may optionally include a DNA base at their 3' end. In some embodiments, two or more adjacent RNA bases may be incorporated into the chimeric primer at the 5' position of the overlap region that would result when an unmodified DNA version of the chimeric primer hybridizes to another primer in the reaction mixture under the conditions selected for a given PCR assay.

[0041] In some embodiments, the present disclosure provides chimeric primers that can be used to reduce or eliminate non-specific amplification artifacts and primer-dimer formation without requiring preliminary testing to first identify problematic primer pairs in a given reaction mixture, which is advantageous in that it can save time and resources.For example, the introduction of an RNA base into the overlap region formed by a pair of primers reduces primer-dimer formation.However, a primer may interact with multiple other primers in a reaction mixture, resulting in different pairings with different overlap regions.Assuming that the overlap region varies from pair to pair, repeated testing may be required to determine the optimal position for the RNA base to be incorporated into a given chimeric primer.In contrast, the method described herein allows the design of chimeric primers based solely on the sequence of DNA primers that can be used in PCR assays.No additional information or testing is required, reducing development time and costs.

[0042] A chimeric primer according to any of the exemplary embodiments disclosed herein may contain an RNA analog instead of one or more of the RNA bases described herein. For example, a chimeric primer may contain at least one segment containing two or more adjacent RNA analogs. An RNA analog may be, for example, 2'-O-methyl RNA, in which a methyl group is added to the 2' hydroxyl of the ribose moiety. Other RNA analogs known in the art may also be used. For brevity, this disclosure generally refers to chimeric primers containing RNA bases. However, for clarity, any such reference is also intended to contemplate alternative embodiments in which one or more of such RNA bases are replaced by an RNA analog.

[0043] Without being bound by theory or mechanism, it is believed that the DNA-RNA bond is more stable, and therefore the chimeric primer is more likely to align only with the DNA template in the PCR reaction mixture, preventing the formation of primer-dimers. The simple primer design rules described herein are amenable to automation and can be used to significantly reduce (if not eliminate) the lengthy iterative design and testing process required by current methods. Thus, the methods described herein can be used to more quickly develop and / or optimize PCR assays, reducing effort, time, cost, and time to market.

[0044] <PCRアッセイ> The present disclosure provides PCR methods using chimeric primers, as well as compositions and kits useful for such methods. In some aspects, the PCR reaction mixture may contain at least one chimeric primer (e.g., two or more chimeric primer pairs). The PCR reaction mixture may also contain PCR reaction reagents, which may be nucleotides such as dGTP, dATP, dTTP, and dCTP, a DNA polymerase such as a thermostable DNA polymerase, and a pH buffer containing salts (e.g., MgCl) and other components necessary for PCR. In certain embodiments, the PCR reaction mixture may further contain a nucleic acid sample (e.g., including genomic DNA and / or mRNA). In certain embodiments, the components of the PCR reaction may be at concentrations suitable for PCR.

[0045] Targeted PCR conditions include those known in the art (e.g., Ausubel, et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons 1995, and Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd third Edition, 2001 Cold Spring Harbor, NY). The amount of amplification product may be assessed after any number of PCR amplifications (i.e., successive cycles of denaturation, renaturation, and polymerization). In certain embodiments, the amount of any amplification product may be assessed during the linear phase of nucleic acid amplification (i.e., during the linear phase of the amplification reaction) or after the reaction rate has reached a plateau.

[0046] When a multiplex PCR mixture containing chimeric primers according to the present disclosure is used in a PCR method, the method can generate at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 50 or more analyzable amplification products. The reaction mixture may be used in a multiplex PCR method to simultaneously amplify 10 or more products, 50 or more products, 100 or more products, 250 or more products, 500 or more products, 1,000 or more products, 2,500 or more products, 5,000 or more products, or 10,000 or more products, in certain cases without detectable (or significant) primer-dimer formation. A multiplex PCR method using chimeric primers according to the present disclosure may be used to amplify the number of target PCR products by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 5-fold, or at least 10-fold compared to an otherwise identical method using only DNA primers.

[0047] The results obtained from the assay may be graphed, and in certain embodiments, the size and / or abundance of the amplification products may be calculated. Similarly, any such products may be sequenced in whole or in part. Any assessment may be qualitative or quantitative. The PCR amplification methods described herein may be performed using a thermal cycler (e.g., a SureCycler 8800 Thermal Cycler by Agilent Technologies, Inc., a Veriti Thermal Cycler by Thermo Fisher Scientific, or a thermal cycler sold by another manufacturer).

[0048] <Chimeric primers and their design methods> A "chimeric primer" according to the present disclosure is an oligonucleotide containing deoxynucleotides and ribonucleotides (also referred to herein as DNA bases and RNA bases, respectively), and has a sequence that includes a segment containing at least two adjacent RNA bases. As understood in the art, a "primer" is an oligonucleotide that can be extended from its 3' end by the action of a polymerase as part of an in vivo or in vitro DNA synthesis reaction. An oligonucleotide that cannot be extended from its 3' end by the action of a polymerase is not a primer. Thus, all chimeric primers must be usable to amplify DNA (e.g., when used in a PCR reaction, as part of a pair of primers that can amplify a segment of genomic DNA).

[0049] In some embodiments, the chimeric primer according to the present disclosure may have a DNA base at its 3' end. The segment comprising at least two adjacent RNA bases may be located anywhere in the chimeric primer. However, in some embodiments, this position is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 bases of the 3' end of the primer. In some embodiments, the chimeric primer may comprise multiple segments, each comprising at least two adjacent RNA bases, and each of these segments is separated from each other by at least two adjacent DNA bases. For example, the chimeric primer may comprise two or three segments, each comprising two adjacent RNA bases, and two adjacent DNA bases separating any of these segments.

[0050] In some embodiments, a chimeric primer may be a primer that functions as part of a pair with a DNA primer, for example, one forward primer and the other reverse primer configured to double a given amplicon. In some embodiments, the primer pair may be configured to double an amplicon while reducing or eliminating primer dimers compared to the amplification product generated when a DNA primer equivalent is used instead of the chimeric primer under otherwise identical PCR assay conditions. In some embodiments, the chimeric primers described herein may be used in PCR assays, including multiplex PCR assays, to generate final products containing less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% of primer dimer amplification products compared to another identical PCR assay using a DNA primer equivalent instead of such a chimeric primer.

[0051] In some embodiments, a chimeric primer may comprise an oligonucleotide in which at least two RNA bases comprise a pair of RNA bases located at positions 7 and 8, or positions 14 and 15, measured from the 3' end of the chimeric primer. In other embodiments, a chimeric primer may comprise a pair of RNA bases located at positions 2 and 3, 3 and 4, 4 and 5, 5 and 6, 6 and 7, 8 and 9, 9 and 10, 10 and 11, 11 and 12, 13 and 14, 15 and 16, 16 and 17, 17 and 18, 18 and 19, 19 and 20, or any combination thereof.

[0052] In some embodiments, chimeric primers capable of amplifying DNA while minimizing or eliminating non-specific amplification may be generated by a method comprising: a) identifying non-specific amplification fragments (e.g., primer dimers) generated during a PCR assay; b) identifying one or more DNA primers that generate the non-specific amplification fragments; and c) selecting a sequence for at least one chimeric primer, wherein the chimeric primer is an oligonucleotide containing DNA and RNA bases and has a sequence identical to that of one of the identified DNA primers, except for a first segment in which at least two adjacent DNA bases are replaced by corresponding RNA bases. In some embodiments, the sequence for the chimeric primer may be selected based on the hybridization of one or more primers that generates non-specific amplification products. For example, a given pair of primers may be known to generate a specific primer dimer. The pair of primers may be analyzed (e.g., by software) to determine the extent to which the primers overlap under the PCR assay conditions and parameters in which they will be used. Once the overlap region is identified, one or more segments of either of the DNA primers may be selected for replacement with RNA bases 5' of the overlap region to generate a chimeric primer. This process may be applied to either or both DNA primers and is illustrated by the examples below. Chimeric primers generated using the methods described herein can be used for standard or multiplex PCR and may be provided, for example, in a kit for performing PCR.

[0053] Incorporation of at least one chimeric primer into a PCR assay typically reduces primer-dimer formation. Figure 1 illustrates the results of a series of PCR assays using standard DNA primers, chimeric primers generated according to Peleg's disclosure, and chimeric primers according to the present disclosure (which, contrary to Peleg's approach, have two adjacent RNA bases). The protocol and parameters of this study are described in more detail below as "Experiment 2." Shaded boxes highlight primer pairs that resulted in primer-dimer formation when at least one of the DNA primers was replaced with a chimeric primer. This data confirms that PCR assays using chimeric primers according to the present disclosure result in much less primer-dimer formation than assays using standard DNA versions of the same primers. Furthermore, the chimeric primers generated according to the present disclosure performed comparable to, and in some cases even better than, Peleg's chimeric primers. This result is surprising and unexpected because the chimeric primers generated according to the present disclosure contain adjacent RNA bases, contrary to Peleg's rule, which prohibits the use of constructs containing adjacent RNA bases.

[0054] Further experiments were performed to examine the effect of incorporating multiple adjacent RNA bases into at least one primer used in a PCR assay. As illustrated by Figure 2, several chimeric primers were generated by replacing one, two, or three adjacent DNA bases in a standard DNA primer with the corresponding RNA bases. These RNA bases were incorporated at the 5' position of the overlap region that occurs when an unmodified primer hybridizes with the other member of its primer pair under standard PCR assay conditions. The protocol and parameters of this study are described in more detail below as "Experiment 3." Shaded columns represent primer pairs tested where at least one primer in the assay was a chimeric primer (i.e., unshaded columns represent unmodified DNA primer pairs). The results of this study indicate that chimeric primers containing two or three RNA bases tend to be more effective than chimeric primers with a single RNA base. This study also demonstrates that suppression of a specific primer pair does not result in other primer pairs forming new or additional primer-dimers, highlighting the utility and applicability of this method for multiplex PCR assays (including several, if not many, primer pairs).

[0055] Figure 3 summarizes the results of another study investigating whether converting DNA primers to chimeric primers affects amplicon yield. The protocol and parameters of this study are described in more detail below as "Experiment 4." In this example, a multiplex PCR assay was performed using various pairs of DNA primers. The amplicon coverage results for this assay are shown in the column titled "pMixDNA." This PCR assay was repeated using several primers converted to chimeric primers according to the present disclosure, and the amplicon coverage results are shown in the column titled "pMix3." Shaded boxes represent primer pairs that include at least one chimeric primer. As illustrated by this figure, the relative coverage of most tested amplicons was not significantly affected by conversion to chimeric primers.

[0056] In general, the use of chimeric primers for PCR amplification increases specificity and reduces the formation of nonspecific fragments, as evidenced by Figures 4 and 5. Figure 4 shows three chromatograms (Genescans) showing the results of a PCR assay performed using a universal DNA primer (top) on genomic DNA and repeated with two different sets of universal chimeric primers (middle and bottom). Although the universal DNA primers should have no complements in the genome, nonspecific fragments were nevertheless formed (i.e., as confirmed by the multiple peaks in the top chromatogram). The middle and bottom chromatograms show no peaks, meaning that no nonspecific fragments were detected, thus confirming the increased specificity of the chimeric primers tested. All chimeric primers tested in this assay contained at least one group of two adjacent RNA bases. Figure 5 is a set of three chromatograms summarizing the results of a related study, comparing amplification products generated by 30 cycles of PCR using universal DNA primers (top) or universal chimeric primers (middle and bottom). Nonspecific fragments were detected only in the products generated by the DNA primers, again confirming the superior performance of the chimeric primers generated by this disclosure over standard DNA primers.

[0057] Figure 6 shows an exemplary workflow for generating chimeric primers according to the present disclosure, highlighting the location of the overlap region in this representative primer pair. This diagram illustrates that the process can begin with identifying a primer-dimer sequence. Once a dimer sequence is identified, the primer pair that gave rise to this primer can be identified by analyzing the primer pair included in the standard or multiplex PCR assay from which the primer-dimer sequence was generated. Primer-dimers form during PCR amplification as a result of unintended hybridization of at least two primers contained in the reaction mixture. Thus, once a set of at least two primers associated with dimer formation has been identified, the method can proceed by identifying the overlap region between at least two primers of the set, as shown in step 3 of this diagram. In some embodiments, this overlap region may be identified by determining the pairwise alignment of selected primer sequences under the conditions used for a given PCR assay (e.g., taking into account the salt concentration, temperature parameters, etc. to be used). The overlap region may also be determined using computational modeling (e.g., using molecular dynamics simulations). After determining the overlap region, a chimeric primer version of either (or both) of the original DNA primers can be generated by selecting at least two adjacent deoxynucleotides 5' of the overlap region and converting these deoxynucleotides to ribonucleotides. In this example, steps 4 and 5 illustrate the selection and conversion of two deoxynucleotides immediately adjacent to the overlap region on both primer sequences. It is understood that any of the steps in this chimeric primer design process (except for the final synthesis of the primer molecules) may be performed using software. In particular, it is envisioned that this method can be included in an automated process to rapidly design and optimize sets of primers for use in multiplex PCR assays.

[0058] In some embodiments, chimeric primers capable of amplifying DNA while minimizing or eliminating nonspecific amplification may be generated by a method comprising: a) selecting a DNA primer from a pair of DNA primers configured to amplify at least a portion of a template DNA molecule in a PCR assay; b) selecting a sequence for the chimeric primer, the chimeric primer being an oligonucleotide comprising DNA and RNA bases, the sequence comprising a first segment consisting of two adjacent RNA bases, the sequence being identical to the sequence of the DNA primer selected in step a) except for the first segment; and c) optionally generating the chimeric primer (e.g., by any known oligonucleotide synthesis method). In some embodiments, the first segment spans positions 3 and 4, 5 and 6, 6 and 7, 7 and 8, 8 and 9, or 14 and 15, measured from the 3' end of the chimeric primer. In other embodiments, the first segment may span any two adjacent bases of the chimeric primer.

[0059] The chimeric primers designed by this method may be used to amplify DNA in a PCR assay. For example, such a method may include: b) performing a PCR assay using a reaction mixture comprising one or more chimeric primers, a DNA-dependent polymerase, and a template DNA molecule; and c) amplifying at least one segment of the template DNA molecule using the one or more chimeric primers, wherein each of the one or more chimeric primers is an oligonucleotide comprising DNA and RNA bases, and has a sequence comprising a first segment comprising at least two adjacent RNA bases.

[0060] Chimeric primers generated according to the present disclosure can be conveniently generated without prior testing to identify problematic primer pairs (e.g., pairs that form primer dimers). Furthermore, as discussed above, primers can interact with multiple other primers in a given reaction mixture, resulting in multiple pairings with different overlap regions. The existence of these multiple pairings complicates the identification of problematic primer pairs and the identification of overlap regions that can be targeted for conversion to RNA bases. Figure 7 illustrates an example of this problem, highlighting the overlap between primers. Primer ADHv3_0265-F is involved in both primer dimers, one overlapping by 10 bp and the other by only 4 bp. Assuming that both primer dimers account for more than 15% of the total primer dimers in a given PCR assay, both should be avoided. However, if chimeric primer design rules requiring information about the overlap region are used, this strategy would require designing and testing two possible chimeric primers for ADHv3_0265-F.

[0061] Choice 1:AAGACTCGGCAGCATCTCCATGTTTACCATrUrUGTTGGCAGAG

[0062] Choice 2: AAGACTCGGCAGCATCTCCATGTTTACCATTTGTTGrGrCAGAG

[0063] To determine which one performs better, both chimeric primers must then be tested in separate PCR assays, which is resource and time consuming. This troubleshooting and refinement process often requires significant resources for large multiplex PCR assays involving numerous primers.

[0064] In contrast, the chimeric primer design rules disclosed herein only require information about the sequence of the initial DNA primer to be converted into a chimeric primer, and therefore can be applied to eliminate the need for iterative testing and refinement processes. A pair of adjacent DNA bases at a specific position according to the DNA primer sequence can be converted into their corresponding RNA bases, thereby reducing the formation of primer-dimers while maintaining amplicon coverage. [Example]

[0065] Example 1: Evaluation of chimeric primers designed without prior information on dimer formation Figure 8 summarizes the results of a study examining the effect of converting DNA bases to RNA bases at five fixed positions, gradually shifted from the 3' end to the 5' end of the chimeric primer. As shown by Figure 8, shifting adjacent pairs of RNA bases more toward the 5' end has an effect on yield.

[0066] Based on these results, new primers were designed in which the RNA bases were positioned at three fixed positions: the third and fourth bases counting from the 3' end ("Version 1"), the seventh and eighth bases counting from the 3' end ("Version 2"), and the fourteenth and fifteenth bases counting from the 3' end ("Version 3"). These chimeric primers were tested in PCR assays to analyze the levels of primer dimers formed by these chimeric primers compared to standard DNA primers (all chimeric primers are represented by shading). The results of this study are summarized in the table shown in Figure 9. As illustrated by Figure 9, moving the RNA base further away from the 3' end (Version 3) still reduced primer dimer amplification, but to a much lower extent than the other two versions. For most of the tested primers, there was no significant difference in the reduction of primer dimer levels between Version 1 and Version 2. Thus, in this exemplary set of primers, chimeric primers with adjacent pairs of RNA bases at positions 3 and 4, or 7 and 8, were shown to be particularly effective at reducing the level of primer-dimer formation.

[0067] The chimeric primers examined in this study were further investigated to analyze amplicon coverage. The coverage of the regular amplicon is not affected by the position of the RNA base. As illustrated by the graph shown in Figure 10, the overall coverage of all primers in the three different primer mixtures is nearly identical. Expanding the coverage of the chimeric primers (Figure 11) reveals that in versions 1 and 2, the coverage of these chimeric primers is somewhat identical, while in version 3, the coverage is slightly higher. Without being bound by theory, it appears that in version 3, the RNA bases are further away from the 3' end, reducing their impact on primer-dimer formation but no longer reducing amplification efficiency, and their impact on alignment and amplification is reduced.

[0068] Multiplex PCR assays are becoming increasingly common, and the chimeric primers disclosed herein are suitable for such assays. To improve the number of samples customers can handle per run, the relative coverage of each amplicon in the assay must be relatively similar. Because not all primers have the same efficiency, their concentrations in the primer mix must be optimized. To do so in an all-DNA assay, the volume (and therefore concentration) of each primer is individually adjusted until a predetermined criterion is met. The same approach can be applied to assays with chimeric primers. To evaluate the use of chimeric primers in a representative multiplex PCR assay, a complete ADHv3 MASTR assay was prepared using two RNA bases, positions 7 and 8 from the 3' end, for all primers. This assay was performed using the original configuration (MP4) without volume modifications and as a second run (MP5) with volume optimization. Figure 12 is a table showing the relative amplicon coverage of a representative set of primers incorporated into the MP4 and MP5 assays. While some primers were unaffected by the concentration change (e.g., ADHv3_0062, ADHv3_0075), others responded very well (e.g., ADHv3_0083, ADHv3_0358). Only a few primers showed no response at all, and these may be redesigned (e.g., ADHv3_0079, ADHv3_0210). Primers with reduced volumes (e.g., ADHv3_0256, ADHv3_0324) also showed reduced coverage. As illustrated by these results, all chimeric primer assays can be optimized in the same way as all standard DNA assays: by simply varying primer concentrations.

[0069] The amplification products generated by the MP4 and MP5 assays were sequenced and analyzed for primer dimer levels. Figure 13 shows the results of primer dimer analysis after sequencing. The first four samples were run with the chimeric primer assay, and the next four samples were run with the DNA primer assay. The reduction in primer dimers when using chimeric primers is evident, with primer dimer levels dropping from >20% with the DNA primer configuration to <1.5% with the chimeric primer configuration. After optimizing primer concentrations, primer dimer analysis of MP5 (Figure 14) shows a slight increase in primer dimer levels compared to MP4, but this level is still much lower than the all-DNA primer mixture (e.g., >20% vs. approximately 2-4%).

[0070] As illustrated by this exemplary data, assays of all-chimeric primers with RNA bases in place by design have a significant positive impact on primer-dimer generation (e.g., a 20-fold reduction in primer-dimer formation). Furthermore, because chimeric primers bind more specifically to their targets, they amplify fewer off-target fragments (e.g., as demonstrated in the "% Mapping" column in the tables shown in Figures 13 and 14). As a result, the chimeric primers and design methods disclosed herein can be used to efficiently design chimeric primers, thereby avoiding the slow and expensive iterative design process required by known methods.

[0071] Example 2: Reduction of primer dimers in multiplex PCR assays using chimeric primers designed based on information about primer dimers The first multiplex PCR amplification assay (designated "RDP135-5-pMixDNA") was performed using a reaction mixture containing genomic DNA, a set of DNA primers configured to amplify multiple amplicons, and a PCR reaction mixture (containing deoxynucleotides, a thermostable DNA polymerase, a pH buffer containing MgCl2, and other components necessary for PCR) and tested against two samples ("s1" and "s2"). This assay served as a control in this study. Specifically, the first PCR reaction contained a set of specific primers (0.5 μM per primer), 250 μM of each dNTP, 1× Titanium Taq buffer, Taq polymerase, and 3.5 mM MgCl2. PCR proceeded for 10 min at 98°C to denature the template, followed by 20 cycles of amplification between 95°C (45 s, denaturation), 60°C (45 s, annealing), and 68°C (2 min, extension), followed by a final shift to 72°C (10 min). A second PCR on these samples included a set of universal PCR primers (0.5 μM per primer) rather than specific primers, but followed the exact same protocol, except that the annealing step was performed at 64°C.

[0072] A second multiplex PCR amplification assay (designated "RDP135-5-pMix3") was performed on the same two samples using the same conditions and parameters, except that some of the DNA primers were replaced with chimeric primers prepared in accordance with the present disclosure. Specifically, both samples were analyzed to identify pairs of DNA primers that resulted in primer dimers, and each of these problematic DNA primers was replaced with a corresponding chimeric primer incorporating a pair of adjacent ribonucleotides 5' of the overlap region that results when the DNA primers hybridize to each other. DNA primers in both sets that were found not to result in primer dimers were left unmodified.

[0073] The amplification products generated by these four PCR samples were recovered and sequenced. The results of this analysis are summarized in Table 1 below.

[0074] [Table 1]

[0075] As illustrated by Table 1, replacement of the problematic DNA primers in the S1 and S2 sets with the corresponding chimeric primers resulted in a significant reduction in the amount of primer dimers in both cases (i.e., from >70% to approximately 1%).

[0076] Example 3: Comparison of DNA amplification using primers designed according to Peleg's rules to DNA amplification using primers designed according to the present disclosure An experiment was conducted to compare the performance of primer designs according to Peleg's design rules (e.g., with multiple non-adjacent ribonucleotide substitutions) to primers designed according to the present disclosure with at least two adjacent ribonucleotide substitutions. The results of this experiment are summarized in the table illustrated by Figure 1.

[0077] In this experiment, PCR amplification was performed using genomic DNA as a template, following the same PCR protocol as above, except for the structure of the primer pair used for amplification. The control group (designated the "pMix DNA" group) included a DNA primer pair (e.g., the pair "HRR_0965_F" and "HRR_0913_F"). The first experimental group used a variant of the control primer pair (designated the "pMix Chimera (Peleg)" group) with at least two non-adjacent ribonucleotides according to the Peleg design rules. The second experimental group used a variant of the control primer pair (designated the "pMix Chimera (adjacent RNA)" group) with at least two adjacent ribonucleotides according to the present disclosure. After the second PCR amplification, the generated products were recovered using the AMPure® bead purification kit. The amplicon library was diluted to 4 nM by spectrophotometric measurement and sequenced using an Illumina® MiSeq system according to the manufacturer's standard protocol.

[0078] This study confirms that PCR assays using chimeric primers according to the present disclosure result in much less primer dimerization than assays using standard DNA versions of the same primers. Furthermore, the chimeric primers generated according to the present disclosure perform comparable to, and in some cases even better than, Peleg's chimeric primers.

[0079] Example 4: Comparative analysis of the effect of the number of adjacent RNA bases used in chimeric primers Experiments were conducted to examine the effect of incorporating multiple adjacent RNA bases into at least one primer used in a PCR assay. As illustrated by Figure 2, chimeric primers were generated by replacing one, two, or three adjacent DNA bases of a standard DNA primer with the corresponding ribonucleotide base. As shown in this figure, the ribonucleotide base was positioned 5' of the overlap region of each tested primer pair. A series of PCR amplifications were performed using the primer pairs shown in Figure 2 under identical conditions except for the primer structure. The PCR assays followed the same protocol as described above in

[0046] .

[0080] The results of this study show that chimeric primers containing two or three RNA bases tend to be more effective than chimeric primers with a single RNA base. This study also demonstrates that suppressing a specific primer pair prevents other primer pairs from forming new or additional primer-dimers.

[0081] Example 5: Amplicon coverage studies A study was conducted to analyze whether chimeric primers according to the present disclosure affect amplicon coverage. In this study, a multiplex PCR assay was performed using a set of multiple pairs of DNA primers (a control group designated "pMix DNA"). The PCR assay followed the same protocol as described above. The PCR assay was repeated using several primers converted to chimeric primers according to the present disclosure (i.e., "pMix3"). To assess the amplicon coverage achieved by the test primer sets, the amplified products were recovered and sequenced. Sequencing confirmed that the relative coverage of most of the test amplicons was not significantly affected by conversion to chimeric primers, as illustrated by the results shown in Figure 3.

[0082] Example 5: Combinatorial assay using chimeric primers As explained above, chimeric primers can be used to reduce or eliminate primer-dimer formation, allowing for the use of a larger panel of primer pairs in the assay. In this experiment, three multiplex PCR assays using chimeric primers were evaluated separately and in combination as pairs. In this example, the chimeric primers had RNA bases replaced at positions 7 and 8 from the 3' end. These combination assays are only feasible because the chimeric primers used in this study result in a significant reduction in primer dimers. Briefly, four samples (50 ng / μl) of the I-0092 DNA panel were analyzed. The assays were performed separately and in combination as described below, following the OnePlex MASTRplus protocol. The volume of the primer mix used in the combination configuration was the same as in the single-configuration assay. Universal PCRs were purified and analyzed on a GeneScan platform. Libraries were prepared and evaluated on a MiSeq platform.

[0083] Two multiplex PCR assays were tested alone and in combination: (1) a CFTR chimeric assay consisting of 275 amplicons with an average length of 220 bp, and (2) a tetrachimeric assay, a 15-amplicon assay with an average length of 152 bp, targeting 15 SNPs and designed for sample confirmation.

[0084] Experiments were configured to run these assays individually and in combination as a single assay. See Table 2 below for the composition of the multiplex PCR reaction. After one round of purification (1 / 1.7 ratio) and a 250-fold dilution, universal PCR was performed. Universal PCR was performed with one round of purification (1 / 1.1 ratio) and analyzed using Applied Biosystems' GeneScan Analysis Software. Reaction concentrations were measured using the DropSense system, and equimolar mixtures of all samples were prepared. The resulting libraries were analyzed using an Illumina MiSeq system.

[0085] [Table 2]

[0086] As illustrated in Figure 15, analysis of universal PCR using GeneScan shows good amplification levels and the absence of primer dimers for both the individual assays (bottom two rows) and the combined assay (top row). Similarly, both the CFTR chimeric and tetrachimeric assays show low primer dimer formation. As standalone assays, the CFTR chimeric assay averaged 0.21%, while the tetrachimeric assay averaged 0.04%. When the two assays were combined, primer dimer formation remained low (0.08%), as illustrated in Figure 16, which shows the combined assay (top row) and the CFTR chimeric assay (middle row) and tetrachimeric assay (bottom row) results as separate reactions. The combined assay achieved 99.92% mapping reads, which is consistent with the single assays. Amplicon coverage in the combined assay was also good, with reduced amplicons in the case of a single tetrachimeric assay. Figure 17 shows the coverage of the tetra-chimera assay compared between the individual and combined assays. Figure 18 shows the coverage of the CFTR chimera assay compared between the individual and combined assays.

[0087] This study demonstrates that multiplex PCR assays using chimeric primers can potentially be combined to form a single, larger-scale assay that provides diagnostic information for multiple conditions or diseases. This type of combined assay is made possible because a significant reduction in primer-dimer formation is observed when chimeric primers according to the present disclosure are used in place of standard DNA primers.

[0088] All statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents as well as equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Accordingly, the scope of the present invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

[0089] All publications and patents cited herein are incorporated by reference to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference, and are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date of the present application and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be separately confirmed.

Claims

1. 1. A method for generating chimeric primers capable of amplifying at least a portion of a template DNA molecule while minimizing or eliminating primer-dimer formation, comprising: a) identifying primer-dimers generated during a PCR assay; b) identifying one or more DNA primers that give rise to said primer-dimers; c) identifying the overlap region that occurs when the one or more DNA primers hybridize during a PCR assay; and d) selecting a sequence for at least one chimeric primer; and e) optionally generating said at least one chimeric primer wherein each chimeric primer is an oligonucleotide comprising DNA and RNA bases and has a sequence identical to that of one of said identified DNA primers except for a first segment in which at least two adjacent DNA bases are replaced by corresponding RNA bases.

2. The method of claim 1 , wherein the first segment is positioned outside the overlap region.

3. The method of claim 1 or 2, wherein the overlap region is determined based on annealing conditions compatible with a PCR assay.

4. 4. The method of claim 1, wherein the first segment is located within 15 bases of the 3' end of the selected sequence, the sequence having a DNA base at its 3' end.

5. 5. The method of claim 1, wherein the sequence selected for the at least one chimeric primer further comprises a second segment comprising at least two adjacent RNA bases that replace corresponding DNA bases in the identified DNA primer, and wherein the first and second segments are separated by at least two DNA bases.

6. 6. The method of claim 5, wherein the first segment and the second segment are located within 15 bases of the 3' end of the sequence of the selected sequence.

7. 6. The method of claim 5, wherein the sequence of the at least one chimeric primer further comprises a third segment comprising at least two adjacent RNA bases that replace corresponding DNA bases in the identified DNA primer, and the second and third segments are separated by at least two DNA bases.

8. selecting a sequence for the at least one chimeric primer selecting a sequence for a first chimeric primer configured to hybridize to an upstream portion of the template DNA molecule; and selecting a sequence for a second chimeric primer configured to hybridize to a downstream portion of said template DNA molecule; wherein the first chimeric primer and the second chimeric primer are capable of amplifying at least a portion of the template DNA molecule by PCR.

9. 9. The method of claim 8, wherein the first chimeric primer and the second chimeric primer each comprise a sequence having a DNA base at its 3' end, and the first segment of each sequence is located within 15 bases of the respective 3' ends.

10. 10. The method of claim 8 or 9, wherein at least one of the first chimeric primer and the second chimeric primer further comprises a second segment comprising at least two adjacent RNA bases, and the first segment and the second segment are separated by at least two DNA bases.

11. 11. The method of claim 10, wherein both the first chimeric primer and the second chimeric primer each further comprise a second segment comprising at least two adjacent RNA bases, and wherein the first segment and the second segment are separated by at least two DNA bases.

12. 12. The method of any one of claims 1 to 11, wherein the at least one chimeric primer is configured to amplify at least a portion of the template DNA molecule while reducing or eliminating the formation of non-specific amplification products.

13. 12. The method of any one of claims 1 to 11, wherein the at least one chimeric primer is configured to amplify at least a portion of the template DNA molecule while reducing or eliminating primer-dimers and / or off-target amplification.

14. the PCR assay a) less than 5% primer dimer amplification products; b) less than 7% primer dimer amplification products; or c) less than 10% primer dimer amplification products 14. The method of any one of claims 1 to 13, which is a multiplex PCR assay producing an end product comprising:

15. 1. A method for generating chimeric primers capable of amplifying DNA while minimizing or eliminating non-specific amplification, comprising: a) identifying non-specific amplified fragments generated during a PCR assay; b) identifying one or more DNA primers that give rise to said non-specifically amplified fragments; and c) selecting a sequence for at least one chimeric primer wherein the chimeric primer is an oligonucleotide comprising DNA and RNA bases and has a sequence identical to that of one of the identified DNA primers except for a first segment in which at least two adjacent DNA bases are replaced by corresponding RNA bases.

16. 16. The method of claim 15, wherein the corresponding RNA base is located within 15 bases of the 3' end of the sequence, and the sequence has a DNA base at its 3' end.

17. 17. The method of claim 15 or 16, wherein the sequence of the chimeric primer further comprises a second segment comprising at least two adjacent DNA bases replaced by corresponding RNA bases, and wherein the first and second segments are separated by at least two DNA bases.

18. The method of any one of claims 1 to 17, wherein at least one of the RNA bases of the chimeric primer is replaced by an RNA analogue.

19. 19. The method of claim 18, wherein the RNA analog is 2'-O-methyl RNA.

20. 1. A method for amplifying DNA, comprising: a) performing a polymerase chain reaction (PCR) assay using a reaction mixture comprising one or more chimeric primers, a DNA-dependent polymerase, and a template DNA molecule; and b) amplifying at least one segment of said template DNA molecule using said one or more chimeric primers; wherein each of the one or more chimeric primers is an oligonucleotide comprising DNA and RNA bases and has a sequence comprising a first segment comprising at least two adjacent RNA bases.

21. 21. The method of claim 20, wherein at least one of the chimeric primers has a sequence consisting of a) a DNA base and b) at least one pair of adjacent RNA bases spanning positions 3 and 4, 7 and 8, and / or 14 and 15, measured from the 3' end of the chimeric primer.

22. 22. The method of claim 20 or 21, wherein the sequence of at least one of the chimeric primers further comprises a second segment comprising at least two adjacent RNA bases, and the first and second segments are separated by at least two DNA bases.

23. 23. The method of claim 22, wherein the sequence of at least one of the chimeric primers further comprises a third segment comprising at least two adjacent RNA bases, the second segment and the third segment being separated by at least two DNA bases.

24. 21. The method of claim 20, wherein the reaction mixture comprises two chimeric primers, each chimeric primer being an oligonucleotide comprising DNA and RNA bases, having a sequence comprising a first segment comprising at least two adjacent RNA bases.

25. 25. The method of claim 24, wherein the first segment of each chimeric primer is located within 15 bases of the 3' end of the sequence of each chimeric primer, and each sequence has a DNA base at its 3' end.

26. 26. The method of claim 24 or 25, wherein the sequence of each chimeric primer further comprises a second segment comprising at least two adjacent RNA bases, and the first and second segments are separated by at least two DNA bases.

27. 27. The method of claim 26, wherein the sequence of each chimeric primer further comprises a third segment comprising at least two adjacent RNA bases, the second segment and the third segment being separated by at least two DNA bases.

28. 28. The method of claim 27, wherein at least one of the first segment, the second segment, and the third segment is located within 15 bases of the 3' end of the sequence of each chimeric primer.

29. the one or more chimeric primers in the reaction mixture a forward primer configured to hybridize to an upstream portion of the template DNA molecule; and a reverse primer configured to hybridize to a downstream portion of the template DNA molecule; 21. The method of claim 20, wherein the forward primer and the reverse primer are chimeric oligonucleotides, each comprising a sequence comprising DNA and RNA bases and a first segment comprising at least two adjacent RNA bases.

30. 30. The method of claim 29, wherein each of the forward primer and the reverse primer comprises a sequence having a DNA base at its 3' end, and the first segment of each sequence is located within 15 bases of the respective 3' ends.

31. 31. The method of claim 29 or 30, wherein at least one of the forward primer and the reverse primer further comprises a second segment comprising at least two adjacent RNA bases, and wherein the first segment and the second segment are separated by at least two DNA bases.

32. 32. The method of Claim 31, wherein both the forward primer and the reverse primer each further comprise a second segment comprising at least two adjacent RNA bases, and wherein the first segment and the second segment are separated by at least two DNA bases.

33. 33. The method of any one of claims 20 to 32, wherein the one or more chimeric primers are configured to amplify at least a portion of the template DNA molecule while reducing or eliminating the formation of non-specific amplification products.

34. 33. The method of any one of Claims 20 to 32, wherein the one or more chimeric primers are configured to amplify at least a portion of the template DNA molecule while reducing or eliminating primer-dimers and / or off-target amplification.

35. the PCR assay a) less than 5% primer dimer amplification products; b) less than 7% primer dimer amplification products; or c) less than 10% primer dimer amplification products 33. The method of any one of claims 20 to 32, which is a multiplex PCR assay that produces an end product comprising:

36. 36. The method of any one of claims 20 to 35, wherein at least one of the chimeric primers has a sequence consisting of a) a DNA base and b) a pair of adjacent RNA bases at positions 3 and 4 measured from the 3' end of the chimeric primer.

37. 36. The method of any one of claims 20 to 35, wherein at least one of the chimeric primers has a sequence consisting of a) a DNA base and b) a pair of adjacent RNA bases at positions 7 and 8 measured from the 3' end of the chimeric primer.

38. 36. The method of any one of claims 20 to 35, wherein at least one of the chimeric primers has a sequence consisting of a) a DNA base and b) a pair of adjacent RNA bases at positions 14 and 15 measured from the 3' end of the chimeric primer.

39. 39. The method of any one of claims 20 to 38, wherein at least one of the RNA bases of the chimeric primer is replaced by an RNA analogue.

40. 40. The method of claim 39, wherein the RNA analog is 2'-O-methyl RNA.

41. 41. A kit for carrying out the method of any one of claims 20 to 40, comprising the one or more chimeric primers and optionally one or more of: a) a DNA-dependent polymerase, b) a buffer compatible with PCR assays, and c) deoxyribonucleotide triphosphates (dNTPs).

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