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 for amplifying a plurality of different target sequences within a sample, comprising:
a) producing at least one hundred different amplified target sequences by amplifying at least one hundred different target sequences within a single amplification reaction mixture, wherein the producing includes contacting the at least one hundred different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions and forming a plurality of double stranded amplified target sequences containing a primer-derived sequence, and wherein at least one of the plurality of target-specific primers and at least one of the primer-derived sequences includes a cleavable group;
b) cleaving the cleavable group of at least one primer-derived sequence of at least one double stranded amplified target sequence, thereby forming a cleaved amplified target sequence;
c) producing one or more double-stranded adapter-ligated amplified target sequences by ligating at least one double-stranded adapter containing an adapter sequence to the cleaved double-stranded amplified target sequence in a blunt-ended ligation reaction; and
d) reamplifying at least one of the double-stranded adapter-ligated amplified target sequences using primers that bind to at least some portion of the adapter sequence;
wherein the method includes use of no more than two target-specific primers to produce any one of the double stranded adapter-ligated amplified target sequences.
2. The method of claim 1 , wherein one or more of the at least one double-stranded adapter cannot hybridize under high stringency to the 20 contiguous nucleotides at the 3′ end or the 5′ end of at least one amplified target sequence in the sample.
3. The method of claim 1 , wherein the reamplifying includes contacting the at least one double-stranded adapter-ligated amplified target sequence with one or more primers including a nucleic acid sequence that is complementary to at least one of the double-stranded adapters, and a polymerase under amplification conditions, thereby producing at least one double-stranded reamplified adapter-ligated amplified target sequence.
4. The method of claim 1 , wherein at least one target-specific primer is at least 70% complementary to at least a portion of a corresponding target sequence in the sample.
5. The method of claim 1 , wherein at least one double-stranded adapter that is ligated to the cleaved end of the at least one of the double-stranded amplified target sequences is susceptible to exonuclease digestion.
6. The method of claim 1 , wherein at least one double-stranded adapter that is ligated to the cleaved end of the at least one of the double-stranded amplified target sequences does not include a protecting group.
7. The method of claim 1 , wherein the ligating includes contacting at least one double-stranded amplified target sequence having a 3′ end and a 5′ end with a ligation reaction mixture including one or more double-stranded adapters and a ligase under ligation conditions, wherein none of the double-stranded adapters in the ligation reaction mixture includes, prior to the ligating, a target-specific sequence.
8. The method of claim 7 , wherein the ligating includes contacting at least one double-stranded amplified target sequence with a ligation reaction mixture including one or more double-stranded adapters and a ligase under ligation conditions, wherein the ligation reaction mixture does not include one or more additional oligonucleotide adapters prior to ligating the one or more double-stranded adapters to at least one double-stranded amplified target sequence.
9. The method of claim 1 , wherein the amplifying further includes a digestion step prior to the ligating, thereby producing a plurality of double-stranded blunt-end amplified target sequences possessing a 5′ phosphate group.