METHODS AND COMPOSITIONS FOR MULTIPLEX PCR
The present invention provides methods, compositions, kits, systems and apparatus that are useful for multiplex PCR of one or more nucleic acids present in a sample. In particular, various target-specific primers are provided that allow for the selective amplification of one or more target sequences. In one aspect, the invention relates to target-specific primers useful for the selective amplification of one or more target sequences associated with cancer or inherited disease. In some aspects, amplified target sequences obtained using the disclosed methods, kits, systems and apparatuses can be used in various downstream processes including nucleic acid sequencing and used to detect the presence of genetic variants.
1 . A method of amplifying a plurality of different target nucleic acid molecules comprising:
contacting a plurality of different target nucleic acid molecules with a pool of target-specific primers having a cleavable group, and a polymerase under synthesizing conditions, and synthesizing a plurality of different target-specific molecules by extending one or more of the pool of target-specific primers having the cleavable group;
digesting a plurality of the different target-specific molecules having the cleavable group of the target-specific primer;
ligating at least one adapter to at least one of the plurality of digested different target-specific molecules; and
producing at least one adapter-ligated amplified target sequence, wherein the method includes using no more than two target-specific primers to produce any one of the adapter-ligated amplified target sequences.
2 . The method of claim 1 , wherein the cleavable group includes a modified nucleobase or modified nucleoside.
3 . The method of claim 1 , wherein the cleavable group includes an apurinic nucleotide.
4 . The method of claim 1 , wherein the cleavable group includes methylguanine, 8-oxo-guanine, xanthine, hypoxanthine, 5,6-dihydrouracil, uracil, 5-methylcytosine, thymine-dimer, 7-methylguanosine, 8-oxo-deoxyguanosine, xanthosine, inosine, dihydrouridine, bromodeoxyuridine, uridine or 5-methylcytidine.
5 . The method of claim 1 , wherein the cleavable group includes a restriction enzyme recognition site.
6 . The method of claim 1 , wherein the method further includes a sequencing step.
7 . The method of claim 6 , wherein the sequencing step includes sequencing a portion of the at least one adapter-ligated amplified target sequence.
8 . The method of claim 7 , further comprising sequencing a portion of the at least one adapter-ligated amplified target sequence on a next-generation sequencing platform.
9 . The method of claim 1 , wherein the method further includes clonally amplifying a portion of the at least one adapter-ligated amplified target sequence.
10 . The method of claim 9 , wherein clonally amplifying a portion of the at least one adapter-ligated target sequence includes emulsion PCR, bridge PCR or isothermal amplification.
11 . The method of claim 1 , wherein the plurality of different target nucleic acid molecules are obtained from cell-free circulating DNA.
12 . The method of claim 11 , wherein the cell-free circulating DNA is isolated from a maternal subject.
13 . The method of claim 1 , wherein the plurality of different target nucleic acid molecules are from RNA.
14 . The method of claim 13 , wherein the method further includes reverse transcribing the RNA into a population of cDNA molecules prior to the contacting.
15 . The method of claim 1 , wherein the method further includes detecting a genetic variant within one or more of the plurality of different target nucleic acid molecules.
16 . The method of claim 1 , wherein the method further includes determining copy number variation within one or more of the plurality of different target nucleic acid molecules.
17 . The method of claim 1 , wherein the method further includes detecting a point mutation, insertion or deletion within one or more of the plurality of different target nucleic acid molecules.
18 . The method of claim 1 , wherein the method further includes detecting a translocation within one or more of the plurality of different target nucleic acid molecules.
19 . The method of claim 1 , wherein the method further includes detecting a genetic rearrangement within one or more of the plurality of different target nucleic acid molecules.
20 . The method of claim 19 , wherein the method includes hybridizing a first target-specific primer having a cleavable group from the pool of target-specific primers having a cleavable group upstream of a genetic rearrangement, and hybridizing a second target-specific primer having a cleavable group from the pool of target-specific primers having a cleavable group downstream of a genetic rearrangement.
21 . The method of claim 1 , wherein the plurality of different target nucleic acid molecules are obtained from genomic DNA in an amount from 1 ng to 200 ng total genomic DNA.
22 . The method of claim 1 , wherein the plurality of different target nucleic acid molecules are obtained from genomic DNA in an amount from 10 ng to 50 ng of total genomic DNA.
23 . The method of claim 1 , wherein the plurality of different target nucleic acid molecules are obtained from genomic DNA in an amount from 1 ng to 10 ng of total genomic DNA.
24 . The method of claim 1 , wherein the method further includes phosphorylating at least one of the digested different target-specific amplicons at the 5′ end prior to the ligating.
25 . A method of amplifying a plurality of different target nucleic acid sequences comprising:
contacting a portion of the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions,
producing a plurality of different amplified target sequences by extending one or more of the plurality of target-specific primers, wherein a plurality of the target-specific primers include a cleavable group and at least one of the plurality of the amplified target sequences includes a primer-derived sequence containing the cleavable group;
cleaving the cleavable group of the primer-derived sequence of the at least one amplified target sequence,
forming a cleaved end of the at least one amplified target sequence; and
ligating at least one adapter to the cleaved end of the at least one amplified target sequence, wherein the at least one adapter is not complementary to 20 contiguous nucleotides of the cleaved end of the at least one amplified target sequence.
26 . The method of claim 25 , wherein the method includes using no more than two target-specific primers from the plurality of target-specific primers to produce any one of the plurality of different amplified target sequences.
27 . The method of claim 25 , wherein the plurality of different target nucleic acid sequences are obtained from 10 ng to 50 ng of total genomic DNA.
28 . The method of claim 25 , wherein the plurality of different target nucleic acid sequences are obtained from 1 ng to 10 ng of total genomic DNA.
29 . The method of claim 25 , wherein the plurality of different target nucleic acid sequences are obtained from cell-free circulating DNA.
30 . The method of claim 29 , wherein the cell-free circulating DNA is isolated from a maternal subject.