Novel primers and uses thereof

Abstract

Compositions, methods, and kits for amplifying nucleic acids are provided.SOLUTION: (a) a loopable primer comprising a target-specific section, an adaptor section and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, or (b) a split primer comprising a first target-specific section, a second target-specific section and an adaptor section located therebetween, or (c) a split primer comprising a first target-specific section, a second target-specific section, a stem-forming section located therebetween and an adaptor section, or a first adaptor section; A primer-containing composition comprising any of the following: a split-loopable primer comprising a second adaptor section and a stem-forming section and a target-specific section positioned therebetween.SELECTED DRAWING: Figure 1

Description

[Background technology]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 617,066, filed January 12, 2018, which is incorporated by reference herein in its entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy has a creation date of December 27, 2018, is named N_022_WO_Ol_SL.txt, and is 3,844 bytes in size.

[0003] Molecular barcodes or indexing sequences used in next-generation sequencing reduce the quantitative bias introduced by duplication by tagging each nucleic acid fragment with a molecular barcode or indexing sequence. Different molecular barcodes or indexing sequences in sequence reads represent different original nucleic acid molecules. By referencing molecular barcodes or indexing sequences, PCR artifacts, such as sequence changes caused by errors due to the absence of polymerase in the original nucleic acid molecule, can be identified and separated from actual polymorphisms / mutations present in the original nucleic acid molecule. Summary of the Invention [Problem to be solved by the invention]

[0004] However, to apply molecular barcodes or indexing sequences to highly multiplex PCR, there is a need to suppress primer dimer formation, avoid barcode resampling, bind non-specific primers, and reduce the formation of primer concatemers. [Means for solving the problem]

[0005] The present invention relates to compositions, methods, and kits for amplifying nucleic acids. In a first aspect, the invention described herein relates to a composition containing primers, the primers being: (a) a loopable primer comprising a target-specific section, an adapter section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure; or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adapter section located between the first and second target-specific sections; or (c) comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, and an adapter section, or comprising a first adapter section and a second a split-loop-capable primer comprising an adapter section, a stem-forming section located between the first adapter section and the second adapter section, and a target-specific section; The primer corresponds to any one of the primers listed above.

[0006] In a second aspect, the invention described herein relates to a method for amplifying a target locus of interest from template DNA, the method comprising: The method includes at least two preliminary amplification cycles, and the primers used in the preliminary amplification cycles are as follows: (a) a loopable primer comprising a target-specific section, an adapter section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure; or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adapter section located between the first and second target-specific sections; or (c) a split-loop-capable primer comprising a first target-specific section, a second target-specific section, and a stem-forming section located between the first and second target-specific sections, or a first adaptor section, a second adaptor section, a stem-forming section located between the first and second adaptor sections, and a target-specific section, wherein each pre-amplification cycle comprises annealing a primer to a template DNA or a pre-amplification product thereof and extending the annealed primer; The primer corresponds to any one of the primers listed above.

[0007] In a third aspect, the invention described herein relates to a kit for amplifying a target locus of interest, wherein the primers provided in the kit are: (a) a loopable primer comprising a target-specific section, an adapter section, and a stem-forming section, wherein the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure; or (b) a split primer comprising a first target-specific section, a second target-specific section, and an adapter section located between the first and second target-specific sections; or (c) a split-loop capable primer comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, or comprising a first adapter section, a second adapter section, a stem-forming section located between the first and second adapter sections, and a target-specific section; The primer corresponds to any one of the primers listed above.

[0008] In a fourth aspect, the invention described herein relates to a method for determining copy number variation at a target locus in a subject, the method comprising: Pre-amplifying a target locus of interest from template DNA using at least two pre-amplification cycles, comprising: (a) one or more loopable primers, each comprising a target-specific section, an adapter section, a molecular indexing section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section, the molecular indexing section, and the 5' portion of the target-specific section, wherein the adapter section comprises a universal adapter sequence for PCR amplification, and the molecular indexing section comprises a molecular indexing sequence; (b) one or more split-loop-capable primers, each comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, a molecular indexing section, and an adapter section, wherein the stem-forming section is capable of hybridizing to a portion of the second target-specific section to form a loop and stem structure comprising the adapter section and the molecular indexing section, wherein the adapter section comprises a universal adapter sequence for PCR amplification, and the molecular indexing section comprises a molecular indexing sequence; or (c) one or more split-loop-capable primers, each comprising a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, a molecular indexing section, and a target-specific section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section, the molecular indexing section, and the 5' portion of the target-specific section, wherein the first and / or second adaptor section comprises a universal adaptor sequence for PCR amplification, and the molecular indexing section comprises a molecular indexing sequence; preamplification using any of the primers amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; sequencing the amplification products and determining copy number variation at the target locus of interest using molecular indexing sequences; a split-loop capable primer, The primers used are any of the following:

[0009] In a fifth aspect, the invention described herein relates to a method for determining fetal aneuploidy, the method comprising: Pre-amplifying a plurality of target loci of interest on one or more chromosomes from cell-free DNA isolated from a maternal blood sample, using at least two pre-amplification cycles, comprising: (a) a plurality of loopable primers, each comprising a target-specific section, an adapter section, a molecular indexing section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section, the molecular indexing section, and the 5' portion of the target-specific section, wherein the adapter section comprises a universal adapter sequence for PCR amplification, and the molecular indexing section comprises a molecular indexing sequence; (b) a plurality of split-loop-capable primers, each comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, a molecular indexing section, and an adapter section, wherein the stem-forming section is hybridizable to a portion of the second target-specific section to form a loop and stem structure comprising the adapter section and the molecular indexing section, and a split-loopable primer, wherein the primer section comprises a universal adapter sequence for PCR amplification and the molecular indexing section comprises a molecular indexing sequence; or (c) a plurality of split-loop-capable primers, each comprising a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, a molecular indexing section, and a target-specific section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section, the molecular indexing section, and the 5' portion of the target-specific section, wherein the first and / or second adaptor section comprises a universal adaptor sequence for PCR amplification, and the molecular indexing section comprises a molecular indexing sequence; preamplification using any of the primers amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; sequencing the amplification products and determining fetal aneuploidy using molecular indexing sequences; Includes:

[0010] In a sixth aspect, the invention described herein relates to a method of multiplex amplification, the method comprising: Pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles, comprising: (a) at least a first loopable primer and a second loopable primer, each of which primers comprises a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section and the 5' portion of the target-specific section, and the adapter section comprises a universal adapter sequence for PCR amplification; (b) at least a first split-loop-capable primer and a second split-loop-capable primer, each of which comprises a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, wherein the stem-forming section is capable of hybridizing to a portion of the second target-specific section to form a loop comprising the stem structure and the adapter section, and the adapter section comprises a universal adapter sequence for PCR amplification; or (c) at least a first split-loop-capable primer and a second split-loop-capable primer, each of which comprises a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section and the 5' portion of the target-specific section, and wherein the first and / or second adaptor sections comprise a universal adaptor sequence for PCR amplification; first and second split-loop capable primers, wherein the first and second primers contain complementary sequences within their target-specific sections such that a primer dimer can form in the absence of protection by the stem-forming section; preamplification using any of the primers amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; Preventing primer dimer formation is particularly useful in PCR tiling (e.g., when amplifying overlapping or tiled target sequences in a single multiplex PCR reaction).

[0011] In a seventh aspect, the invention described herein relates to a method of allele-specific amplification, the method comprising: Pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles, comprising: (a) a loopable primer comprising a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section and the 5' portion of the target-specific section, the adapter section comprising a universal adapter sequence for PCR amplification, and the loopable primer comprises an SNV or SNP allele in the 5' portion or the 3' portion of the target-specific section; (b) a split-loop-capable primer comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, wherein the stem-forming section is capable of hybridizing to a portion of the second target-specific section to form a loop and stem structure comprising the adapter section, and the adapter section comprises a universal adapter sequence for PCR amplification, and wherein the split-loop-capable primer comprises an SNV or SNP allele in the target-specific section; or (c) a split-loop-capable primer comprising a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section and the 5' portion of the target-specific section, wherein the first and / or second adaptor section comprises a universal adaptor sequence for PCR amplification, and the split-loop-capable primer comprises an SNV or SNP allele in the 3' portion of the target-specific section; preamplification using any of the primers amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; Includes:

[0012] In an eighth aspect, the invention described herein relates to a method of allele-specific quantitative PCR (qPCR), the method comprising: Pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles, comprising: (a) at least a first loopable primer and a second loopable primer, each comprising a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section and the 5' portion of the target-specific section, and wherein the adapter section of the first loopable primer is a universal primer for PCR amplification; a first loopable primer and a second loopable primer, the first loopable primer comprising a universal adapter sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, the adapter section of the second loopable primer comprising a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, the 5' or 3' portion of the target-specific section of the first loopable primer comprising a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second loopable primer comprising a second SNV or SNP allele; (b) at least a first split-loop-capable primer and a second split-loop-capable primer, each comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, and an adapter section, wherein the stem-forming section is capable of hybridizing to a portion of the second target-specific section to form a loop and stem structure comprising the adapter section, and wherein the adapter section of the first split-loop-capable primer comprises a universal adapter sequence for PCR amplification and and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adapter section of the second split-loop-capable primer comprises a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' or 3' portion of the target-specific section of the first split-loop-capable primer comprises a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second split-loop-capable primer comprises a second SNV or SNP allele; or (c) at least a first split-loop-capable primer and a second split-loop-capable primer, each comprising a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section and the 5' portion of the target-specific section, and the first and / or second adaptor sections of the first split-loop-capable primer are capable of performing a PCR amplifying the first and second loop-able primers, wherein the first and / or second adapter section of the second split-loop-able primer comprises a universal adapter sequence for PCR amplification and a second probe-specific sequence that can bind to a second fluorescent probe, the 5' or 3' portion of the target-specific section of the first split-loop-able primer comprising a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second split-loop-able primer comprising a second SNV or SNP allele; amplifying the preamplification product using one or more PCR primers capable of hybridizing to the universal adapter sequence in the presence of the first fluorescent probe and the second fluorescent probe; Detecting real-time intensities of fluorescent signals from the first fluorescent probe and the second fluorescent probe; Includes: Alternatively, the method for allele-specific qPCR does not require a pre-amplification step, but instead involves amplifying one or more target loci of interest from template DNA using primers (a), (b), or (c) in the presence of a first fluorescent probe and a second fluorescent probe; Detecting real-time intensities of fluorescent signals from the first fluorescent probe and the second fluorescent probe; Includes:

[0013] In a ninth aspect, the invention described herein relates to a method of allele-specific digital PCR (dPCR), the method comprising: Pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles, comprising: (a) at least a first loopable primer and a second loopable primer, each comprising a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, and the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the adapter section and the 5' portion of the target-specific section, wherein the adapter section of the first loopable primer comprises a universal adapter sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, and the adapter section of the second loopable primer comprises a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' or 3' portion of the target-specific section of the first loopable primer comprises a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second loopable primer comprises a second SNV or SNP allele; (b) at least a first split-loop-capable primer and a second split-loop-capable primer, each comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, and an adapter section, wherein the stem-forming section is capable of hybridizing to a portion of the second target-specific section to form a loop and stem structure comprising the adapter section, and wherein the adapter section of the first split-loop-capable primer comprises a universal adapter sequence for PCR amplification and and a first probe-specific sequence capable of binding to a first fluorescent probe, wherein the adapter section of the second split-loop-capable primer comprises a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, wherein the 5' or 3' portion of the target-specific section of the first split-loop-capable primer comprises a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second split-loop-capable primer comprises a second SNV or SNP allele; or (c) at least a first split-loop-capable primer and a second split-loop-capable primer, each comprising a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being capable of hybridizing to the 3' portion of the target-specific section to form a loop and stem structure comprising the second adaptor section and the 5' portion of the target-specific section, and the first and / or second adaptor sections of the first split-loop-capable primer are capable of performing a PCR amplifying the first and second loop-able primers, wherein the first and / or second adapter section of the second split-loop-able primer comprises a universal adapter sequence for PCR amplification and a second probe-specific sequence that can bind to a second fluorescent probe, the 5' or 3' portion of the target-specific section of the first split-loop-able primer comprising a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second split-loop-able primer comprising a second SNV or SNP allele; Partitioning the preamplification products into multiple reaction volumes; In the presence of the first fluorescent probe and the second fluorescent probe, amplifying the preamplification product in each reaction volume using one or more PCR primers hybridizable to the sequence; detecting the presence or absence of a fluorescent signal from the first fluorescent probe and the second fluorescent probe; Includes: Alternatively, the method for allele-specific dPCR does not require a pre-amplification step, but instead involves: Partitioning the sample into multiple reaction volumes; amplifying one or more target loci of interest from template DNA using primers (a), (b), or (c) in the presence of a first fluorescent probe and a second fluorescent probe; detecting the presence or absence of a fluorescent signal from the first fluorescent probe and the second fluorescent probe; Includes:

[0014] These and other features, as well as their organization and method of operation, will become apparent from the following detailed description when taken in conjunction with the accompanying drawings. [Brief explanation of the drawings]

[0015] [Figure 1] 1 shows several embodiments of the primers described herein (Schemes AC, and a control scheme). [Figure 2] 1 shows an embodiment of a loopable primer configured such that the 3′ end portion of the target-specific section forms a stem structure with a complementary sequence. [Figure 3] 1 shows an embodiment of a loopable primer configured such that the 5′ end portion of the target-specific section forms a stem structure with a complementary sequence. [Figure 4] 1 shows one embodiment of splitting parameters in which the target-specific section is split into a 5' end portion and a 3' end portion, which are separated by an adapter sequence. [Figure 5] 1 shows an example workflow of an amplification process including two pre-amplification cycles using the primers described herein. [Figure 6] 1 shows the target success rate of an exemplary amplification process involving two pre-amplification cycles using the primers described herein. [Figure 7] 1 shows an example workflow of an amplification process including 3 or 10 pre-amplification cycles using the primers described herein. [Figure 8]1 shows target success rates for exemplary amplification processes involving 3 or 10 pre-amplification cycles using primers described herein. [Figure 9] The MIT counts are shown to be consistent between replicate samples (Scheme A, two pre-amplification cycles). [Figure 10] 1 shows the primer and product sequences according to one embodiment of a loopable primer, including two mismatched nucleotides (nt) of the loopable primer. [Figure 11] 1 shows primer and product sequences according to one embodiment of a loopable primer. [Figure 12] 1 shows primer and product sequences according to one embodiment of a split primer. [Figure 13] 1A-E show several embodiments (Schemes D-E) of the split-loopable primers described herein. [Figure 14] This shows that MIT counts were consistent among replicate samples in highly multiplexed PCR (Scheme A, two preamplification cycles, workflow in Figure 5). The target success rate of the amplification process in this example is 83%. DETAILED DESCRIPTION OF THE INVENTION

[0016] Some of the inventions conceived by the inventors for the purpose of carrying out the invention Reference will now be made in detail to specific embodiments, certain specific examples of which are illustrated in the accompanying drawings. While the invention will be described in conjunction with these specific embodiments, it is understood that it is not intended to limit the invention to the described embodiments. On the contrary, it is intended to cover alternatives, modifications, and equivalents as may be included within the spirit and scope of the invention as defined by the appended claims.

[0017] Although numerous specific details are set forth in the following description to provide a thorough understanding of the present invention, certain exemplary embodiments of the present invention may be implemented without some or all of these specific details.

[0018] The various techniques and mechanisms of the present invention are sometimes described in the singular for clarity, although it should be noted that some embodiments include multiple repetitions of a technique or multiple instantiations of a mechanism unless otherwise specified.

[0019] The disclosures of patent applications incorporated by reference herein are as follows: International Patent Application No. PCT / US2006 / 045281, entitled "SYSTEM AND METHOD FOR CLEANING NOISY GENETIC DATA AND USING DATA TO MAKE PREDICTIONS," International Patent Application No. PCT / US2008 / 003547, entitled "SYSTEM AND METHOD FOR CLEANING NOISY GENETIC DATA AND DETERMINING CHROMSOME COPY NUMBER," International Patent Application No. PCT / US2009 / 034506, entitled "METHODS FOR CELL GENOTYPING," International Patent Application No. PCT / US2009 / 045335, entitled "METHODS FOR EMBRYO CHARACTERIZATION AND COMPARISON," and International Patent Application No. PCT / US2009 / 045335, entitled "METHODS FOR ALLELE CALLING AND PLOIDY International Patent Application No. PCT / US2009 / 052730 entitled "METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING", International Patent Application No. PCT / US2010 / 050824 entitled "METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING", International Patent Application No. PCT / US2011 / 037018 entitled "METHODS FOR NON-INVASIVE PRENATAL PLOIDY CALLING", International Patent Application No. PCT / US2011 / 061506 entitled "METHODS FOR NON-INVASIVE PRENATAL PTERNITY TESTING", International Patent Application No. PCT / US2011 / 066938 entitled "METHODS FOR NON-INVASIVE PRENATAL PTERNITY TESTING", International Patent Application PCT / US Patent Application Publication No. 2012 / 066339, entitled "METHODS AND COMPOSITIONS FOR REDUCING GENETIC COMPOSITIONS"International Patent Application PCT / US Publication No. 2013 / 055205, entitled "METHODS FOR INCREASING FETAL FRACTION IN MATERNAL BLOOD," International Patent Application PCT / US Publication No. 2013 / 057924, entitled "METHODS OF USING LOW FETAL FRACTION DETECTION," International Patent Application PCT / US Publication No. 2014 / 051926, entitled "METHODS OF USING LOW FETAL FRACTION DETECTION," International Patent Application PCT / US Publication No. 2014 / 057843, entitled "PRENATAL DIAGNOSTIC RESTING STANDARDS," and "DETECTING MUTATIONS AND PLOIDY IN CHROMOSOMAL SEGMENTS." ", International Patent Application PCT / US Publication No. 2015 / 026957, entitled "METHODS AND COMPOSITIONS FOR DETERMINING PLOIDY," International Patent Application PCT / US Publication No. 2016 / 031686, entitled "METHODS AND COMPOSITIONS FOR DETERMINING PLOIDY," and U.S. Patent Application No. 15 / 372,279, entitled "COMPOSITIONS AND METHODS FOR IDENTIFYING NULCEIC ACID MOLECULES."

[0020] Loopable Primers

[0021] Many of the embodiments of the invention described herein relate to loopable primers comprising a target-specific section, an adapter section, and a stem-forming section, where the stem-forming section is hybridizable to a portion of the target-specific section to form a stem structure, and the target-specific section is hybridizable to a target sequence of a template DNA for amplification.

[0022] In some embodiments, an adapter section is located between the target-specific section and the stem-forming section, and hybridization between the stem-forming section and a portion of the target-specific section forms a loop containing the adapter section. The adapter section may be located, for example, 5' to the target-specific section and 3' to the stem-forming section. In some embodiments, the adapter section includes a universal adapter sequence for PCR amplification and / or sequencing.

[0023] In some embodiments, the loopable primer further comprises a molecular indexing section containing a molecular indexing sequence. Molecular indexing sequences, molecular index tags (MITs), or unique identifier (UID) sequences are described in Kinde et al., PNAS 108(23):9530-9535 (2011) and U.S. Patent No. 15 / 372,279, entitled "COMPOSITIONS AND METHODS FOR IDENTIFYING NULCEIC ACID MOLECULES," each of which is incorporated by reference in its entirety. In some embodiments, the length of each molecular indexing sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp. When both the forward loopable primer and the reverse loopable primer according to the present invention described herein are used to amplify a target locus of interest, the amplification product may contain two molecular indexing sequences. This combination of molecular indexing sequences allows for even more accurate molecule counting than using a single molecular indexing sequence. In one embodiment, the molecular indexing sequence on each loopable primer is a unique molecular indexing sequence. In another embodiment, the combination of molecular indexing sequences on each pair of loopable forward and reverse primers is unique.

[0024] The molecular indexing section may be located, for example, between the target-specific section and the adapter section. The molecular indexing section may be located, for example, 5' to the target-specific section and 3' to the adapter section. In some embodiments, hybridization between the stem-forming section and a portion of the target-specific section forms a loop comprising the adapter section and the molecular indexing section.

[0025] In some embodiments, the loopable primers described herein exclude primers in which the target-specific section does not form part of the stem structure.

[0026] The loopable primers described herein can significantly improve assay specificity by suppressing primer dimer formation and non-specific binding in highly multiplex PCR because they mask / protect at least part of the universal adapter sequence and molecular indexing sequence, as well as the target-specific sequence.

[0027] Scheme A: One scheme of a loopable primer is shown in Figure 2. In this scheme, the target-specific section includes a 5' portion and a 3' portion, and the stem-forming section is hybridizable to the 3' portion of the target-specific section to form a stem structure. When the 3' portion of the target-specific section is protected within the stem structure, the 5' portion of the target-specific section becomes available for initiation of target hybridization.

[0028] In some embodiments, hybridization between the stem-forming section and the 3' portion of the target-specific section forms a loop comprising the adapter section and the 5' portion of the target-specific section. The stem loop is designed to protect the molecular indexing sequence and the adapter sequence from spurious interactions. The 3'-terminal stem also prevents the primer from extending non-target-specifically (i.e., from forming a dimer of the primer).

[0029] In some embodiments, the loopable primer further comprises one or more mismatched nucleotides at the 3' end of the target-specific section that cannot hybridize to the stem-forming section. In some embodiments, the loopable primer comprises one, two, three, four, or five mismatched nucleotides at the 3' end to prevent A-tailing. Alternatively or additionally, the 5' end of the loopable primer may comprise one or more mismatched nucleotides that cannot hybridize to the target-specific section. In some embodiments, the loopable primer comprises one, two, three, four, or five mismatched nucleotides at the 5' end.

[0030] As illustrated in Figure 10, a preferred embodiment of a loopable primer according to Scheme A comprises, from 5' to 3', one or more mismatched nucleotides, a stem-forming section, an adapter section, a molecular indexing section, and a target-specific section, wherein the stem-forming section is the reverse complement of the 3' portion of the target-specific section.

[0031] In some embodiments, the size of the stem structure formed between the stem-forming section and the 3' portion of the target-specific section is about 5 to 20 bp, or about 5 to 10 bp, or about 10 to 15 bp, or about 15 to 20 bp.

[0032] In some embodiments, the loopable primer according to Scheme A has a preferred annealing temperature and melting temperature suitable for PCR reactions, and the stem-forming section and the 3' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. In some embodiments, the annealing temperature is 60°C or less, 59°C or less, or 58°C or less, or 57°C or less, or 56°C or less, or 55°C or less, 54°C or less, or 53°C or less, or 52°C or less, or 51°C or less, or 50°C or less. In some embodiments, the melting temperature is 60°C or more, or 61°C or more, or 62°C or more, or 63°C or more, or 64°C or more, or 65°C or more, 66°C or more, or 67°C or more, or 68°C or more, or 69°C or more, or 70°C or more. In some embodiments, extreme annealing temperatures, such as 30°C to 80°C, are useful. In some cases, this may be the case.

[0033] Scheme B: Another scheme of a loopable primer is shown in Figure 3. In this scheme, the target-specific section includes a 5' portion and a 3' portion, and the stem-forming section is hybridizable to the 5' portion of the target-specific section to form a stem structure. When the 5' portion of the target-specific section is protected within the stem structure, the 3' portion of the target-specific section becomes available for initiating target hybridization.

[0034] In some embodiments, the loop formed by hybridization between the stem-forming section and the 5' portion of the target-specific section includes the adapter section but not the 3' end portion of the target-specific section. The stem-loop is designed to protect the molecular indexing sequence and the adapter sequence from spurious interactions.

[0035] In some embodiments, the loopable primer further comprises one or more G or C nucleotides located 5' to the target-specific section, with the stem structure stabilized by one or more complementary G / C nucleotides located 3' to the stem-forming section. In some embodiments, the loopable primer comprises 1, 2, 3, 4, or 5 G / C nucleotides located 5' to the target-specific section (i.e., at the neck of the stem-loop).

[0036] As illustrated in FIG. 11, a preferred embodiment of a loopable primer according to Scheme B comprises, from 5' to 3', a stem-forming section, one or more G / C nucleotides, an adapter section, a molecular indexing section, one or more G / C nucleotides, and a target-specific section, wherein the stem-forming section is the reverse complement of the 3' portion of the target-specific section.

[0037] In some embodiments, the size of the stem structure formed between the stem-forming section and the 5' portion of the target-specific section is about 5 to 20 bp, or about 5 to 10 bp, or about 10 to 15 bp, or about 15 to 20 bp.

[0038] In some embodiments, the loopable primer according to Scheme B has preferred annealing and melting temperatures suitable for PCR reactions, and the stem-forming section and the 5' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. In some embodiments, the annealing temperature is preferably 60°C or lower, 59°C or lower, or 58°C or lower, or 57°C or lower, or 56°C or lower, or 55°C or lower, 54°C or lower, or 53°C or lower, or 52°C or lower, or 51°C or lower, or 50°C or lower. In some embodiments, the melting temperature is 60°C or higher, or 61°C or higher, or 62°C or higher, or 63°C or higher, or 64°C or higher, or 65°C or higher, 66°C or higher, or 67°C or higher, or 68°C or higher, or 69°C or higher, or 70°C or higher. In some embodiments, extreme annealing temperatures, such as 30°C to 80°C, may be useful.

[0039] Split Primer

[0040] Many of the embodiments of the invention described herein relate to split primers that include a first target-specific section, a second target-specific section, and an adapter section located between the first and second target-specific sections, where the target-specific sections are hybridizable to a target sequence of a template DNA for amplification. do.

[0041] In some embodiments, the split primer further includes a molecular indexing section containing a molecular indexing sequence. The molecular indexing section may be located, for example, between the adapter section and one of the target-specific sections. The molecular indexing section may be located, for example, 3' of the adapter section. In some embodiments, the length of each molecular indexing sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp. When both the forward split primer and the reverse split primer described herein are used to amplify a target locus of interest, the amplification product may contain two molecular indexing sequences. This combination of molecular indexing sequences allows for even more accurate molecular counting than using a single molecular indexing sequence. In one embodiment, the molecular indexing sequence on each split primer is a unique molecular indexing sequence. In another embodiment, the combination of molecular indexing sequences on each pair of split forward and reverse primers is made unique.

[0042] In some embodiments, the adapter section includes a universal adapter sequence for PCR amplification and / or sequencing.

[0043] Scheme C: One scheme of a split primer is shown in Figure 4. In this scheme, an adapter section is located between a first target-specific section and a second target-specific section, both of which are available for hybridization to the target sequence.

[0044] In other words, the target-specific section is split into two parts, with a universal adapter sequence between them. After both ends of the primer bind to the target sequence, the molecular indexing sequence and the adapter sequence are protected. Split primers can be advantageous in that they shorten the sequencing distance.

[0045] As illustrated in Figure 12, a preferred embodiment of a split primer according to Scheme C comprises, from 5' to 3', a first target-specific section, an adapter section, a molecular indexing section, and a second target-specific section.

[0046] Split-Loop Primer

[0047] Many of the embodiments of the invention described herein relate to split-loop capable primers, which primers comprise: (a) comprising a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, or (b) comprising a first adapter section, a second adapter section, a stem-forming section located between the first and second adapter sections, and a target-specific section, wherein the (first and / or second) target-specific section is capable of hybridizing to a target sequence of a template DNA to be amplified.

[0048] In some embodiments, the split-loopable primer further comprises a molecular indexing section containing a molecular indexing sequence, for example, located between the adapter section and the (second) target-specific section. The molecular indexing section may be located, for example, 3' to the (second) adapter section. In some embodiments, the length of each molecular indexing sequence is about 1-20 bp, or about 2-15 bp, or about 3-10 bp, or about 4-8 bp. When both a forward split-loop-capable primer and a reverse split-loop-capable primer according to the present invention described herein are used to amplify a target locus of interest, the amplification product may contain two molecular indexing sequences. This combination of molecular indexing sequences allows for even more accurate molecular counting than using a single molecular indexing sequence. In one embodiment, the molecular indexing sequence on each split-loop-capable primer is a unique molecular indexing sequence. In another embodiment, the combination of molecular indexing sequences on each pair of split-loop-capable forward and reverse primers is unique.

[0049] In some embodiments, the (first and second) adapter sections include universal adapter sequences for PCR amplification and / or sequencing.

[0050] Scheme D: Figure 13 shows one scheme of a split-loop-capable primer. This scheme includes a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, where the stem-forming section is hybridizable to a 5' portion of the target-specific section to form a stem structure. When the first target-specific section is protected within the stem structure, the second target-specific section becomes available for initiation of target hybridization.

[0051] In some embodiments, hybridization between the stem-forming section and the second target-specific section forms a loop comprising the adapter section and the molecular indexing sequence. The stem loop is designed to protect the molecular indexing sequence and the adapter sequence from spurious interactions. The 3'-end stem also prevents the primer from extending non-target-specifically (i.e., from forming a dimer of the primer).

[0052] In some embodiments, the split-loop-capable primer further comprises one or more mismatched nucleotides at the 3'-end of the second target-specific section that cannot hybridize to the stem-forming section. In some embodiments, the split-loop-capable primer comprises one, two, three, four, or five mismatched nucleotides at the 3'-end to prevent A-tailing. Alternatively or additionally, the 5'-end of the stem-forming section may comprise one or more mismatched nucleotides that cannot hybridize to the second target-specific section. In some embodiments, the split-loop-capable primer comprises one, two, three, four, or five mismatched nucleotides at the 5'-end of the stem-forming section.

[0053] As illustrated in Figure 13, a preferred embodiment of a split-loop-capable primer according to Scheme D comprises, from 5' to 3', a first target-specific section, one or more mismatched nucleotides, a stem-forming section, an adapter section, a molecular indexing section, and a second target-specific section, wherein the stem-forming section is the reverse complement of a portion of the second target-specific section.

[0054] In some embodiments, the size of the stem structure formed between the stem-forming section and the second target-specific section is about 5-20 bp, or about 5-10 bp, or is about 10 to 15 bp, or about 15 to 20 bp.

[0055] In some embodiments, the first target-specific section is longer than the second target-specific section, hi some embodiments, the second target-specific section is longer than the first target-specific section.

[0056] In some embodiments, at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90% of the second target-specific section is capable of hybridizing to the stem-forming region to form a stem.

[0057] In some embodiments, the split-loop-capable primer according to Scheme D has a preferred annealing temperature and melting temperature suitable for a PCR reaction, and the stem-forming section and second target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. In some embodiments, the annealing temperature is preferably 60°C or lower, 59°C or lower, or 58°C or lower, or 57°C or lower, or 56°C or lower, or 55°C or lower, 54°C or lower, or 53°C or lower, or 52°C or lower, or 51°C or lower, or 50°C or lower. In some embodiments, the melting temperature is 60°C or higher, or 61°C or higher, or 62°C or higher, or 63°C or higher, or 64°C or higher, or 65°C or higher, 66°C or higher, or 67°C or higher, or 68°C or higher, or 69°C or higher, or 70°C or higher. In some embodiments, extreme annealing temperatures, such as 30°C to 80°C, may be useful.

[0058] Scheme E: 13 shows another scheme of a split-loop-capable primer. In this scheme, the primer comprises a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section comprising a 5' portion, wherein the stem-forming section is hybridizable to a 3' portion of the target-specific section to form a stem structure. When the 3' portion of the target-specific section is protected within the stem structure, the 5' portion of the target-specific section becomes available for initiating target hybridization.

[0059] In some embodiments, the loop formed by hybridization between the stem-forming section and the 3' portion of the target-specific section comprises the second adapter section, the molecular indexing sequence, and the 5' portion of the target-specific section. The stem loop is designed to protect the molecular indexing sequence and the second adapter sequence from spurious interactions. The stem at the 3' end also prevents the primer from extending in a non-target-specific manner (i.e., from forming a dimer).

[0060] In some embodiments, the split-loop-capable primer further comprises one or more mismatched nucleotides at the 3' end of the target-specific section that cannot hybridize to the stem-forming section. In some embodiments, the split-loop-capable primer comprises one, two, three, four, or five mismatched nucleotides at the 3' end to prevent A-tailing. Alternatively or additionally, the 5' end of the stem-forming section may comprise one or more mismatched nucleotides that cannot hybridize to the target-specific section. In some embodiments, the split-loop-capable primer comprises one, two, three, four, or five mismatched nucleotides at the 5' end of the stem-forming section.

[0061] As illustrated in Figure 13, a preferred embodiment of a split-loop-capable primer according to Scheme A comprises, from 5' to 3', a first adapter section, one or more mismatched nucleotides, a stem-forming section, a second adapter section, a molecular indexing section, and a target-specific section, wherein the stem-forming section is the reverse complement of the 3' portion of the target-specific section.

[0062] In some embodiments, the size of the stem structure formed between the stem-forming section and the 3' portion of the target-specific section is about 5 to 20 bp, or about 5 to 10 bp, or about 10 to 15 bp, or about 15 to 20 bp.

[0063] In some embodiments, the first adaptor section is longer than the second adaptor section, hi some embodiments, the second adaptor section is longer than the first adaptor section.

[0064] In some embodiments, the split-loop-capable primer according to Scheme E has a preferred annealing temperature and melting temperature suitable for PCR reactions, and the stem-forming section and the 3' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. In some embodiments, the annealing temperature is preferably 60°C or lower, 59°C or lower, or 58°C or lower, or 57°C or lower, or 56°C or lower, or 55°C or lower, 54°C or lower, or 53°C or lower, or 52°C or lower, or 51°C or lower, or 50°C or lower. In some embodiments, the melting temperature is 60°C or higher, or 61°C or higher, or 62°C or higher, or 63°C or higher, or 64°C or higher, or 65°C or higher, 66°C or higher, or 67°C or higher, or 68°C or higher, or 69°C or higher, or 70°C or higher. In some embodiments, extreme annealing temperatures, such as 30°C to 80°C, may be useful.

[0065] Primer composition

[0066] Further embodiments of the invention described herein relate to primer compositions comprising the loopable primers, split primers and / or split-loopable primers described herein.

[0067] In some embodiments, the primer composition comprises at least a forward loopable primer and a reverse loopable primer, which target the same locus of interest for amplification. In some embodiments, both the forward loopable primer and the reverse loopable primer correspond to Scheme A shown in Figure 2. In some embodiments, both the forward loopable primer and the reverse loopable primer correspond to Scheme B illustrated in FIG.

[0068] In some embodiments, the primer composition comprises at least a forward split primer and a reverse split primer, which target the same locus of interest for amplification. In some embodiments, both the forward split primer and the reverse loopable split primer correspond to Scheme C shown in Figure 3.

[0069] In some embodiments, the primer composition comprises at least a forward split-loopable primer and a reverse split-loopable primer, which target the same locus of interest for amplification. , both the forward split-loopable primer and the reverse split-loopable primer correspond to Scheme E illustrated in FIG. 13. In some embodiments, both the forward split-loopable primer and the reverse split-loopable primer correspond to Scheme E illustrated in FIG.

[0070] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of forward and reverse loopable primers.

[0071] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers, each comprising a different stem-forming section. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers, each comprising a different molecular indexing sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 loopable primers, each comprising a different combination of stem-forming section and molecular indexing sequence.

[0072] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of forward and reverse loopable split primers.

[0073] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split primers, each comprising a different target-specific section. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split primers, each comprising a different molecular indexing sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 split primers, each comprising a different combination of target-specific section and molecular indexing sequence.

[0074] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-loopable primers. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of forward and reverse split-loopable primers.

[0075] In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-loopable primers, each comprising a different stem-forming section. In some embodiments, the composition comprises at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-loopable primers, each comprising a different molecular indexing sequence. In some embodiments, the composition comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 split-loop capable primers, each comprising a different combination of stem-forming section and molecular indexing sequence.

[0076] Nucleic acid amplification method

[0077] Further embodiments of the invention described herein include The present invention relates to a method for amplifying a target locus of interest from template DNA, the method comprising: The method includes at least two pre-amplification cycles using the loopable primer or the split primer or the split-loopable primer, each pre-amplification cycle including annealing a primer to the template DNA or its pre-amplification product and extending the annealed primer.

[0078] In some embodiments, the method comprises: At least 3, at least 4, at least 5, at least 10, or up to 15, or up to 10, or up to 7, or up to 5 pre-amplification cycles.

[0079] In some embodiments, each pre-amplification cycle comprises annealing at least a forward loopable primer and a reverse loopable primer that target the same locus of interest to the template DNA or its pre-amplification product, and extending the annealed forward loopable primer and the annealed reverse loopable primer, in some embodiments, both the forward loopable primer and the reverse loopable primer correspond to Scheme A shown in Figure 2. In some embodiments, both the forward loopable primer and the reverse loopable primer correspond to Scheme B illustrated in FIG.

[0080] In some embodiments, each pre-amplification cycle comprises annealing at least a forward split primer and a reverse split primer that target the same locus of interest to the template DNA or its pre-amplification product, and extending the annealed forward split primer and the annealed reverse split primer. In some embodiments, both the forward loopable primer and the reverse loopable primer correspond to Scheme C illustrated in FIG.

[0081] In some embodiments, each pre-amplification cycle comprises annealing at least a forward split-loopable primer and a reverse split-loopable primer that target the same locus of interest to the template DNA or its pre-amplification product, and extending the annealed forward split-loopable primer and the annealed reverse split-loopable primer, in some embodiments, both the forward split-loopable primer and the reverse split-loopable primer correspond to Scheme D shown in Figure 13. In some embodiments, both the forward split-loopable primer and the reverse split-loopable primer correspond to Scheme E illustrated in FIG.

[0082] As illustrated in FIG. 10, when a pair of forward and reverse loopable primers according to Scheme A is used, the preliminary amplification product may include, from 5' to 3', one or more mismatched nucleotides, a first stem-forming section, a first adapter section, a first molecular indexing section, an amplified target sequence, a second molecular indexing section, a second adapter section, a second stem-forming section, and one or more mismatched nucleotides.

[0083] As illustrated in FIG. 11, when a pair of forward and reverse loopable primers according to Scheme B is used, the preliminary amplification product may include, from 5' to 3', a first stem-forming section, one or more G / C nucleotides, a first adapter section, a first molecular indexing section, an amplified target sequence, a second molecular indexing section, a second adapter section, one or more G / C nucleotides, and a second stem-forming section.

[0084] As illustrated in Figure 12, when a pair of forward and reverse split primers according to Scheme C is used, the preliminary amplification product may include, from 5' to 3', a 5' target-specific sequence, a first adapter section, a first molecular indexing section, an amplified target sequence, a second molecular indexing section, a second adapter section, a second stem-forming section, and a 3' target-specific sequence.

[0085] In some embodiments, the adapter section comprises a universal adapter sequence for PCR amplification, further comprising multiple PCR cycles using one or more PCR primers hybridizable to the universal adapter sequence.

[0086] In some embodiments, the PCR primers comprise sequence adapters for downstream high-throughput sequencing of the PCR products, hi some embodiments, the PCR primers comprise sample barcodes for pooling the PCR products for further analysis.

[0087] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers, each primer comprising a different stem-forming section, to the template DNA or its pre-amplification product. In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers. In some embodiments, each pre-amplification cycle comprises annealing at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different loop-capable primers, each primer comprising a different combination of stem-forming section and molecular indexing sequence, to the template DNA or its pre-amplification products.

[0088] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different loopable primers, each comprising a pair of forward and reverse loopable primers, to the template DNA or its pre-amplification products.

[0089] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split primers, each primer comprising a different target-specific section, to the template DNA or its pre-amplification products. In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split primers, each primer comprising a different molecular indexing sequence, to the template DNA or its pre-amplification products. In some embodiments, each pre-amplification cycle comprises annealing at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different split primers, each primer comprising a different combination of target-specific section and molecular indexing sequence, to the template DNA or its pre-amplification product.

[0090] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different pairs of forward and reverse loopable split primers to the template DNA or its pre-amplification products.

[0091] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-loop-capable primers, each primer comprising a different stem-forming section, to the template DNA or its pre-amplification products. In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different split-loop-capable primers, each primer comprising a different molecular indexing sequence, to the template DNA or its pre-amplification products. In some embodiments, each pre-amplification cycle comprises at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, at least 10,000, or at least 10,000 nucleotides. The method includes annealing at least 10,000, at least 20,000, at least 50,000, or at least 100,000 different split-loop capable primers, each primer containing a different combination of stem-forming section and molecular indexing sequence, to the template DNA or a preliminary amplification product thereof.

[0092] In some embodiments, each pre-amplification cycle comprises annealing at least 50, at least 100, at least 200, at least 500, at least 1,000, at least 2,000, at least 5,000, or at least 10,000 different forward and reverse split-loop-capable primer pairs to the template DNA or its pre-amplification products.

[0093] In some embodiments, the loopable primer according to Scheme A has a preferred annealing temperature and melting temperature suitable for a PCR reaction, and the stem-forming section and the 3' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. The annealing temperature of this pre-amplification cycle is below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0094] In some embodiments, the loopable primer according to Scheme B has a preferred annealing temperature and melting temperature suitable for a PCR reaction, and the stem-forming section and the 5' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. The annealing temperature of this pre-amplification cycle is below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0095] In some embodiments, the split-loop-capable primer according to Scheme C has a preferred annealing temperature and melting temperature suitable for a PCR reaction, and the stem-forming section and the second target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. The annealing temperature of this pre-amplification cycle is below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0096] In some embodiments, the split-loop-capable primer according to Scheme E has a preferred annealing temperature and annealing temperature suitable for a PCR reaction, and the stem-forming section and the 5' portion of the target-specific section form a stem structure at or below the preferred annealing temperature, but do not form a stem structure at or above the melting temperature. The annealing temperature for this pre-amplification cycle is below the preferred annealing temperature (e.g., 60°C or below, 59°C or below, or 58°C or below, or 57°C or below, or 56°C or below, or 55°C or below, 54°C or below, or 53°C or below, or 52°C or below, or 51°C or below, or 50°C or below).

[0097] In other embodiments, the annealing temperature of the pre-amplification cycle may be below 50° C., or below 40° C., or below 30° C., or below 20° C., or above 60° C., or above 65° C., or above 70° C. In other embodiments, the extreme annealing temperatures are: It may be useful for a preliminary amplification cycle such as 30°C to 80°C.

[0098] Nucleic acid amplification kits

[0099] A further embodiment of the invention described herein relates to a kit for amplifying a target locus of interest from template DNA, the kit comprising the loopable primer described above or the split primer described above or the split-loopable primer described above.

[0100] In some embodiments, the kit comprises at least a forward loopable primer and a reverse loopable primer, which target the same locus of interest for amplification. In some embodiments, the kit comprises at least a forward split primer and a reverse split primer, which target the same locus of interest for amplification. In some embodiments, the kit comprises at least a forward split loopable primer and a reverse split loopable primer, which target the same locus of interest for amplification.

[0101] In some embodiments, the kit further comprises a polymerase for extending the loopable primer or split primer or split-loopable primer during the pre-amplification cycle.

[0102] In some embodiments, the kit further comprises a protease for inactivating the polymerase upon completion of the pre-amplification cycle.

[0103] In some embodiments, the kit further comprises one or more PCR primers capable of hybridizing to a universal adapter sequence of the adapter section of the loopable primer, split primer, or split-loopable primer. In some embodiments, the PCR primers comprise sequence adapters for downstream high-throughput sequencing of the PCR products. In some embodiments, the PCR primers comprise a sample barcode for pooling the PCR products for further analysis.

[0104] Purpose

[0105] The loopable primer of Scheme A (3' target stem-loop) conceals / protects the universal adapter sequence and MIT sequence, suppressing primer dimer formation and nonspecific binding in highly multiplexed PCR, thereby improving assay specificity. Therefore, applications in which the loopable primer of Scheme A is particularly useful include the following:

[0106] Detection of copy number variation (CNV), i.e., aneuploidies, microdeletions, etc.: In situations where each DNA fragment product is attached to a unique (or unique combination of) molecular index tags, tracking of the number of fragments in a sample at a particular locus (sequence of the amplicon) becomes possible.

[0107] Elimination of PCR errors, detection of actual mutations: PCR artifacts, e.g., sequence changes caused by polymerase errors not present in the original molecule, can be identified by using MIT barcodes and separated from actual polymorphisms / mutations present in the original molecule.

[0108] PCR tiling: The 3' stem of the primer prevents primer dimerization This is extremely useful for applications where overlapping or tiled amplicons are amplified in a single multiplex PCR reaction.

[0109] Allele-specific amplification: Allele-specific primers with mutant bases located in the stem region (3' end of the primer) inhibit the stem from opening in the mismatched wild type, thereby preventing amplification of the wild type.

[0110] Mutant allele-specific quantitative PCR (qPCR) and digital PCR (qPCR): Mutant and wild-type primers have different tag sequences, which allow for detection by various fluorescent probe colors and other detection methods.

[0111] Additional embodiments of the invention described herein relate to methods for determining copy number variation at a target locus in a subject, the methods comprising: preamplifying a target locus of interest from template DNA using at least two preamplification cycles with one or more loopable primers, each of the loopable primers comprising a target-specific section, an adapter section, a molecular indexing section, and a stem-forming section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being hybridizable to the 3' portion of the target-specific section to form a stem structure, the adapter section comprising a universal adapter sequence for PCR amplification, and the molecular indexing section comprising a molecular indexing sequence; amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; sequencing the amplification products and using molecular indexing sequences to determine copy number variation at the target locus of interest; Includes:

[0112] Additional embodiments of the invention described herein relate to methods of determining fetal aneuploidy, the methods comprising: preamplifying a plurality of target loci of interest on one or more chromosomes from cell-free DNA isolated from a maternal blood sample using at least two preamplification cycles with a plurality of loopable primers, each of the loopable primers comprising a target-specific section, an adapter section, a molecular indexing section, and a stem-forming section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being hybridizable to the 3' portion of the target-specific section to form a stem structure, the adapter section comprising a universal adapter sequence for PCR amplification, and the molecular indexing section comprising a molecular indexing sequence; amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; sequencing the amplification products and determining fetal aneuploidy using molecular indexing sequences; Includes:

[0113] Additional embodiments of the invention described herein relate to a method of multiplex amplification, the method comprising: preamplifying one or more target loci of interest from template DNA using at least two preamplification cycles with at least a first loopable primer and a second loopable primer, each loopable primer comprising a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section is preamplifying the primer, the primer comprising a 5' portion and a 3' portion, the stem-forming section being capable of hybridizing to the 3' portion of the target-specific section to form a stem structure, the adapter section comprising a universal adapter sequence for PCR amplification, the first loopable primer and the second loopable primer comprising complementary sequences within their target-specific sections such that a primer dimer can form in the absence of protection by the stem-forming section; amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; Includes:

[0114] Additional embodiments of the invention described herein relate to a method of allele-specific amplification, the method comprising: preamplifying one or more target loci of interest from template DNA using at least two preamplification cycles with a loopable primer, wherein the loopable primer comprises a target-specific section, an adapter section, and a stem-forming section, wherein the target-specific section comprises a 5' portion and a 3' portion, the stem-forming section is capable of hybridizing to the 3' portion of the target-specific section to form a stem structure, the adapter section comprises a universal adapter sequence for PCR amplification, and the loopable primer comprises an SNV or SNP allele in the 5' portion or the 3' portion of the target-specific section; amplifying the preamplification product using one or more PCR primers hybridizable to the universal adapter sequence; Includes:

[0115] An additional embodiment of the invention described herein relates to a method of allele-specific quantitative PCR (qPCR), the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles with at least a first loopable primer and a second loopable primer, each of the loopable primers comprising a target-specific section, an adapter section, and a stem-forming section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being capable of hybridizing to the 3' portion of the target-specific section to form a stem structure, the adapter section of the first loopable primer comprising a universal adapter sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, the adapter section of the second loopable primer comprising a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, the 5' or 3' portion of the target-specific section of the first loopable primer comprising a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second loopable primer comprising a second SNV or SNP allele; amplifying the preamplification product using one or more PCR primers capable of hybridizing to the universal adapter sequence in the presence of a first fluorescent probe and a second fluorescent probe; Detecting real-time intensities of fluorescent signals from the first fluorescent probe and the second fluorescent probe; Includes: Alternatively, the method for allele-specific qPCR does not require a pre-amplification step, but instead amplifying one or more target loci of interest from template DNA using first and second loopable primers in the presence of first and second fluorescent probes; detecting real-time intensities of fluorescent signals from the first and second fluorescent probes; Includes:

[0116] An additional embodiment of the invention described herein relates to a method of allele-specific quantitative PCR (qPCR), the method comprising: pre-amplifying one or more target loci of interest from template DNA using at least two pre-amplification cycles with at least a first loopable primer and a second loopable primer, each of the loopable primers comprising a target-specific section, an adapter section, and a stem-forming section, the target-specific section comprising a 5' portion and a 3' portion, the stem-forming section being capable of hybridizing to the 3' portion of the target-specific section to form a stem structure, the adapter section of the first loopable primer comprising a universal adapter sequence for PCR amplification and a first probe-specific sequence capable of binding to a first fluorescent probe, the adapter section of the second loopable primer comprising a universal adapter sequence for PCR amplification and a second probe-specific sequence capable of binding to a second fluorescent probe, the 5' or 3' portion of the target-specific section of the first loopable primer comprising a first SNV or SNP allele, and the 5' or 3' portion of the target-specific section of the second loopable primer comprising a second SNV or SNP allele; Partitioning the preamplification products into multiple reaction volumes; amplifying the preamplification product in each reaction volume using one or more PCR primers hybridizable to the universal adapter sequence in the presence of a first fluorescent probe and a second fluorescent probe; detecting the presence or absence of a fluorescent signal from the first fluorescent probe and the second fluorescent probe; Includes: Alternatively, the method for allele-specific dPCR does not require a pre-amplification step, but instead involves: Partitioning the sample into multiple reaction volumes; amplifying one or more target loci of interest from template DNA in each reaction volume using first and second loopable primers in the presence of first and second fluorescent probes; detecting the presence or absence of a fluorescent signal from the first and second fluorescent probes; Includes: [Example]

[0117] Example 1 Two-cycle workflow

[0118] Proof-of-concept experiments were performed to amplify sample DNA using a two-cycle workflow, as illustrated in Figure 5. For each primer scheme combination (Figure 1), a commercially available high-fidelity enzyme mix and DNA were prepared. [Table 1]

[0119] The conditions for cycling the MIT:sample by direct PCR reaction were as follows: After two cycles, 20 μL of prepared protease solution was added to the reaction mixture, which was then incubated at 65°C for 15 minutes and then inactivated at 95°C for 15 minutes. [Table 2] [Table 3]

[0120] Sequencing of barcoding reactions: 10 μL of the resulting 30 μL volume was taken into a Q5 barcoding reaction and cycled 35 times to allow for a complete plateau. [Table 4] [Table 5]

[0121] Storage and purification: 2 μL of each sample was pooled together and a 50 μL pool was purified using a Qiagen Qiaquick spin column. [Table 6]

[0122] Samples were quantified by qPCR and sequenced.

[0123] As shown in Figure 6, Scheme A, Scheme B, and Scheme C each amplified the target locus of interest, or over two preliminary amplification cycles (followed by downstream PCR amplification using primers hybridizable to universal adapter sequences). Scheme A demonstrated the best target success rate. As shown in Figure 9, MIT counts were highly consistent between replicate samples (Scheme A, two preliminary amplification cycles).

[0124] Example 2 3 / 10 cycle workflow

[0125] Proof-of-concept experiments were performed using either a 3-cycle or 10-cycle workflow, as illustrated in Figure 7, to amplify sample DNA. For each combination of primer schemes (Figure 1), a commercially available high-fidelity enzyme mix and DNA were prepared. [Table 7]

[0126] MIT by direct PCR reaction: Samples were cycled under the following conditions: after 3 or 10 cycles, 20 μL of prepared protease solution was added to the reaction, which was then incubated at 65°C for 15 minutes and then inactivated at 95°C for 15 minutes. [Table 8] [Table 9]

[0127] Sequencing of barcoding reactions: A 10 μL volume of the resulting 30 μL was taken up in a Q5 barcoding reaction and cycled 35 times to a complete plateau. [Table 10] [Table 11]

[0128] Storage and purification: 2 μL of each sample was pooled together and a 50 μL pool was purified using a Qiagen Qiaquick spin column. [Table 12]

[0129] Samples were quantified by qPCR and sequenced.

[0130] As shown in Figure 8, Scheme A, Scheme B, and Scheme C were each able to amplify the target locus of interest over three or ten pre-amplification cycles (followed by downstream PCR amplification using primers hybridizable to the universal adapter sequences). Scheme A showed the best target success rate over three pre-amplification cycles, and Scheme C showed the best target success rate over ten pre-amplification cycles.

[0131] In light of the foregoing description, it will be readily apparent to one skilled in the art that various substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein can be practiced without the use of any element or elements or limitation(ies) not specifically disclosed herein. The terms and expressions used are used as terms of description rather than limitation. In using such terms and expressions, there is no intention to exclude the features shown and described or equivalents thereof, but the invention It is recognized that various modifications may be made within the scope of the invention. Thus, it will be understood that, while the present invention has been exemplified by specific embodiments and optional features, modifications and / or variations of the concepts disclosed herein may occur to those skilled in the art, and such modifications and variations are deemed to be within the scope of the present invention.

Claims

1. 1. A composition comprising at least 50 split-loopable primers, each of the split-loopable primers comprising: The above composition comprises a first target-specific section, a second target-specific section, a stem-forming section located between the first and second target-specific sections, and an adapter section, or comprises a first adapter section, a second adapter section, a stem-forming section located between the first and second adapter sections, and a target-specific section.

2. 1. A composition, wherein the split-loop capable primer comprises a first target-specific section, a second target-specific section, a stem-forming section located between the first target-specific section and the second target-specific section, and an adapter section, 2. The composition of claim 1, wherein the composition comprises at least a forward split-loopable primer and a reverse split-loopable primer that target the same locus of interest for amplification.

3. The composition of claim 2, wherein the adapter section is located between the stem-forming section and the second target-specific section, and hybridization of the stem-forming section with a portion of the second target-specific section forms a loop including the adapter section.

4. The composition of claim 3, wherein the adapter section is located 5' to the second target-specific section and 3' to the stem-forming section, and preferably the split-loop-capable primer further comprises one or more mismatched nucleotides at the 3' end of the second target-specific section that cannot hybridize to the stem-forming section.

5. The composition of any one of claims 2 to 4, wherein the split-loopable primer further comprises a molecular indexing section comprising a molecular indexing sequence, preferably the molecular indexing section is located between the adapter section and the second target-specific section.

6. The composition of claim 5, wherein the molecular indexing section is located 5' to the second target-specific section and 3' to the adapter section, and hybridization of the stem-forming section with a portion of the second target-specific section forms a loop comprising the adapter section and the molecular indexing section.

7. the split-loop-capable primer comprises a first adaptor section, a second adaptor section, a stem-forming section located between the first adaptor section and the second adaptor section, and a target-specific section; 2. The composition of claim 1, wherein the composition preferably comprises at least a forward split-loopable primer and a reverse split-loopable primer that target the same locus of interest for amplification.

8. a second adaptor section is located between the stem-forming section and the target-specific section, and hybridization of the stem-forming section with a portion of the target-specific section forms a loop comprising the second adaptor section, The composition of claim 7, wherein the second adapter section is preferably located 5' to the target-specific section and 3' to the stem-forming section.

9. The composition of claim 7 or 8, wherein the split-loop-capable primer further comprises a molecular indexing section comprising a molecular indexing sequence, preferably the molecular indexing section being located between the second adapter section and the target-specific section.

10. The composition of claim 9, wherein the molecular indexing section is located 5' to the target-specific section and 3' to the second adapter section, and hybridization of the stem-forming section with a portion of the target-specific section forms a loop comprising the second adapter section and the molecular indexing section.

11. a target-specific section comprising a 5' portion and a 3' portion, a stem-forming section capable of hybridizing to the 3' portion of the target-specific section, and hybridization of the stem-forming section to the 3' portion of the target-specific section forms a loop comprising a second adaptor section and the 5' portion of the target-specific section, Preferably, the split-loop-capable primer further comprises one or more mismatched nucleotides at the 3' end of the target-specific section that are not hybridizable to the stem-forming section.

12. The composition of any one of claims 1 to 11, wherein the adapter section comprises a universal adapter sequence for PCR amplification and / or sequencing.

13. 13. A composition according to any one of claims 1 to 12, wherein at least 50 split-loopable primers each comprise a different stem-forming section and / or a different molecular indexing sequence, preferably the composition comprises at least 2,500 different primers each comprising a different combination of stem-forming section and molecular indexing sequence.

14. 1. A method for amplifying a target locus of interest from template DNA, comprising at least two pre-amplification cycles using at least 50 split-loop-capable primers, each of which comprises a first target-specific section, a second target-specific section, and a stem-forming section located between the first and second target-specific sections, or a first adaptor section, a second adaptor section, and a stem-forming section located between the first and second adaptor sections; The above method, wherein each pre-amplification cycle comprises annealing a primer to the template DNA or its pre-amplification product and extending the annealed primer.

15. 1. A kit for amplifying a target locus of interest comprising at least 50 split-loopable primers, each of the split-loopable primers comprising: The above kit comprises a first target-specific section, a second target-specific section, and a stem-forming section located between the first target-specific section and the second target-specific section, or comprises a first adaptor section, a second adaptor section, and a stem-forming section located between the first adaptor section and the second adaptor section.

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