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, comprising:
amplifying a plurality of different target sequences within a single amplification reaction mixture, wherein the amplifying includes contacting the plurality of different target sequences with a plurality of target-specific primers including a cleavable group and with a polymerase under amplification conditions and producing a plurality of amplified target sequences containing a primer-derived sequence at one end, and wherein at least one of the target sequences of the plurality is amplified using one pair of target-specific primers having minimal cross-hybridization with any other primer in the amplification reaction mixture, wherein the five terminal nucleotides at the 3′ end of each of the target-specific primers of the one pair do not overlap with the five terminal nucleotides at the 3′ end of any other primer in the amplification reaction mixture, and wherein at least one of the target-specific primers and the primer-derived sequence of at least one of the amplified target sequences includes a cleavable group;
cleaving the cleavable group of a primer-derived sequence of at least one of the amplified target sequence, thereby producing a cleaved amplified target sequence; and
producing at least one adapter-ligated amplified target sequence by ligating at least one double-stranded adapter to an end of at least one cleaved amplified target sequence, wherein the at least one double-stranded adapter is not complementary to the at least one amplified target sequence.
2. The method of claim 1 , wherein the cleavable group includes uracil.
3. The method of claim 1 , further including performing the cleaving prior to the ligating.
4. The method of claim 1 , wherein one or more of the at least one double-stranded adapter is less than 15% complementary to at least one amplified target sequence.
5. The method of claim 1 , wherein at least one of the target-specific primers is less than 15% complementary to any sequence of the sample other than its corresponding target sequence.
6. A method for synthesizing two or more different target sequences within a sample including a plurality of target sequences, comprising:
a) producing a plurality of different synthesized target sequences by contacting a plurality of different target sequences with a plurality of target-specific primers and a polymerase under polymerizing conditions in a single reaction vessel, wherein at least one of the target-specific primers includes a cleavable group and is complementary across its entire length to a corresponding target sequence, and wherein at least one of the resulting synthesized target sequences includes a primer derived sequence containing a cleavable group;
b) cleaving a cleavable group of the primer-derived sequence of the at least one synthesized target sequence and producing a synthesized target sequence including a cleaved primer-derived sequence at one end; and
c) ligating a double-stranded adapter to an end of at least two of the plurality of synthesized target sequences, thereby producing at least two double-stranded adapter-ligated synthesized target sequences, wherein the double-stranded adapter sequence is not complementary to the at least two synthesized target sequences.
7. The method of claim 6 , further including resynthesizing at least one of the double-stranded adapter-ligated synthesized target sequences, wherein the resynthesizing includes contacting at least one of the double-stranded adapter-ligated synthesized target sequences with a primer and a polymerase under polymerizing conditions, thereby producing a plurality of resynthesized adapter-ligated synthesized target sequences.
8. The method of claim 6 , wherein the at least one double-stranded adapter includes a universal primer sequence.
9. The method of claim 1 , wherein minimal cross-hybridization is performed under stringent hybridization conditions.
10. The method of claim 1 , wherein the amplifying includes amplifying a starting input of about 10 ng of nucleic acid.
11. The method of claim 1 , wherein the amplifying includes amplifying a starting input of about 1 ng of nucleic acid.
12. The method of claim 1 , further including sequencing at least a portion of at least one adapter-ligated amplified target sequence.
13. The method of claim 6 , further including sequencing at least a portion of at least one of the adapter-ligated synthesized target sequences.
14. The method of claim 6 , wherein the cleavable group includes uracil.
15. The method of claim 6 , wherein the double stranded adapter is not complementary to 20 contiguous nucleotides from a 3′ end or 5′ end of the at least two synthesized target sequences.
16. The method of claim 1 , wherein the at least one double-stranded adapter is not complementary to 20 contiguous nucleotides from a 3′ end or 5′ end of the at least one amplified target sequence.