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 generating an amplicon library from genomic DNA comprising:
contacting a portion of a genomic DNA with a plurality of different target-specific primers having a cleavable group, and a polymerase under amplification conditions and producing a plurality of different target-specific amplicons by extending one or more of the plurality of target-specific primers having a cleavable group;
digesting at least one of the plurality of different target-specific amplicons with a cleaving reagent; and
ligating an adapter to the end of the at least one digested different target-specific amplicon and producing at least one adapter-ligated target-specific amplicon, wherein the adapter is not complementary to 20 contiguous nucleotides from a 3′ end or 5′ end of the at least one digested different target-specific amplicon.
2 . The method of claim 1 , wherein the genomic DNA is from 1 ng to 200 ng of total genomic DNA.
3 . The method of claim 1 , wherein the genomic DNA is from 10 ng to 50 ng of total genomic DNA.
4 . The method of claim 1 , wherein the genomic DNA is from 1 ng to 10 ng of total genomic DNA.
5 . 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.
6 . The method of claim 1 , wherein the cleavable group includes a modified nucleobase or modified nucleoside.
7 . The method of claim 1 , wherein the cleavable group includes an apurinic nucleotide.
8 . 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.
9 . The method of claim 1 , wherein the cleavable group includes a restriction enzyme recognition site.
10 . The method of claim 1 , wherein the method further includes a sequencing step.
11 . The method of claim 10 , wherein the sequencing step includes sequencing a portion of the at least one adapter-ligated target-specific amplicon.
12 . The method of claim 1 , wherein the method further includes clonally amplifying a portion of the at least one adapter-ligated target-specific amplicon.
13 . The method of 12 , wherein clonally amplifying a portion of the at least one adapter-ligated target-specific amplicon includes emulsion PCR, bridge PCR or isothermal amplification.
14 . The method of claim 1 , wherein the genomic DNA is obtained from cell-free circulating DNA.
15 . The method of claim 14 , wherein the cell-free circulating DNA is isolated from a maternal subject.
16 . The method of claim 1 , wherein the method further includes detecting a genetic variant within at least one of the adapter-ligated target-specific amplicons.
17 . The method of claim 1 , wherein the method further includes determining copy number variation within at least one of the adapter-ligated target-specific amplicons.
18 . The method of claim 1 , wherein the method further includes detecting a point mutation, insertion or deletion within at least one of the adapter-ligated target-specific amplicons.
19 . The method of claim 1 , wherein the method further includes detecting a translocation within at least one of the adapter-ligated target-specific amplicons.
20 . The method of claim 1 , wherein the method further includes detecting a genetic rearrangement within at least one of the adapter-ligated target-specific amplicons.
21 . The method of claim 1 , wherein the adapter is a single-stranded or double stranded adapter.
22 . The method of claim 21 , wherein the adapter further includes a barcode, tag or universal priming sequence.
23 . The method of claim 21 , wherein the double stranded adapter is ligated to the end of at least one digested different target specific amplicon using a ligase capable of blunt-ended ligation.
24 . The method of claim 23 , wherein the ligase is an isothermal ligase.
25 . The method of claim 1 , wherein the genomic DNA is isolated from a single cell.