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. An in vitro cell free composition comprising at least 50 target-specific in vitro amplified primer products each having at least one cleavable group, a polymerase, a cleaving reagent capable of cleaving the at least one cleavable group from a plurality of the at least 50 target-specific in vitro amplified primer products, a ligase capable of blunt-ended ligation, and a plurality of target molecules; wherein the composition is prepared directly from a sample and not from a portion of the sample pre-enriched for target molecules;
wherein each of the target specific primer regions of the primer products have the following criteria:
(1) includes two or more modified nucleotides within the primer sequence, at least one of which is included near or at the termini of the primer and at least one of which is included at, or about the center nucleotide position of the primer sequence;
(2) length is about 15 to about 40 bases in length;
(3) T m is from about 60° C. to about 70° C.;
(4) has low cross-reactivity with non-target sequences present in the sample; (5) at least the first four nucleotides (going from 3′ to 5′ direction) are non-complementary to any sequence within any other primer present in the reaction; and
(6) are non-complementary to any consecutive stretch of at least 5 nucleotides within any other produced amplified target sequence.
2. The composition of claim 1 , wherein the plurality of target molecules is between 1-10 ng of genomic DNA or cDNA.
3. The composition of claim 1 , wherein the ligase capable of blunt-ended ligation is an isothermal ligase.
4. The composition of claim 1 , wherein the ligase capable of blunt-ended ligation is not a thermostable ligase.
5. The composition of claim 1 , wherein the ligase capable of blunt-ended ligation is an ATP dependent ligase.
6. The composition of claim 1 , further comprising dNTPs.
7. The composition of claim 1 , further comprising a plurality of adapters.
8. The composition of claim 7 , further comprising a plurality of double-stranded adapters.
9. The composition of claim 7 , wherein at least one of the plurality of adapters further includes a barcode, a tag, or universal priming sequence.
10. The composition of claim 8 , wherein the plurality of double-stranded adapters do not include a nucleic acid sequence that is complementary to the terminal 10 nucleotides at the 5′ end of the at least 50 target-specific extended primer products or a nucleic acid sequence that is complementary to the terminal 10 nucleotides at the 3′ end of the at least 50 target-specific extended primer products.
11. The composition of claim 7 , wherein the plurality of adapters do not include a phosphorylated adapter.
12. The composition of claim 1 , further comprising one or more DNA polymerase antibodies capable of recognizing a DNA polymerase.
13. The composition of claim 1 , wherein the polymerase includes a proofreading activity.
14. The composition of claim 1 , wherein the plurality of target molecules are present in an amount from 1 ng to 200 ng.
15. The composition of claim 1 , wherein the plurality of target molecules are present in an amount from 10 ng to 50 ng.
16. The composition of claim 1 , wherein the plurality of target molecules are present in an amount less than 1 ng.
17. The composition of claim 1 , wherein the plurality of target molecules are isolated from a single cell.
18. The composition of claim 1 , wherein a portion of the at least 50 target-specific amplified primer products having at least one cleavable group are complementary to a portion of at least one gene selected from the group consisting of EGFR, FLT3, KRAS, BRAS, NRAS and TP53.
19. The composition of claim 1 , wherein a portion of the at least 50 target-specific amplified primer products having at least one cleavable group are complementary to a portion of at least one gene selected from the group consisting of FLT3, BRCA1, BRCA2, ALK, ROS1, RET or CFTR.
20. The composition of claim 1 , wherein at least one amplified primer product is obtained using a primer pair, wherein the primer pair includes a forward primer and a reverse primer, wherein the forward primer hybridizes to a first intron and the reverse primer hybridizes to a second intron, and wherein the primer pair amplifies an exon between the first and second intron.