Methods and compositions for multiplex PCR

Patent metadata

Field	Value
Publication number	US-9951380-B2
Application number	US-201514795774-A
Country	US
Kind code	B2
Filing date	Jul 9, 2015
Priority date	Apr 28, 2011
Publication date	Apr 24, 2018
Grant date	Apr 24, 2018

Abstract

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.

First claim

We claim:

1. A method of generating an amplicon library from genomic DNA comprising:
a) contacting a portion of a nucleic acid sample with a plurality of different target-specific primers, 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;
b) digesting at least one of the plurality of different target-specific primers with a cleaving reagent; and
c) ligating an adapter to the end of the at least one different target-specific amplicon and producing at least one adapter-ligated target-specific amplicon;

wherein each of the plurality of target specific primers have the following criteria:
(1) includes two or more modified cleavable 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; and

wherein none of the adapters in a ligation mixture, prior to ligating, can hybridize under high stringency, to any one of the target specific amplicon sequences in the reaction; and
wherein the method is optionally carried out in a single reaction, addition only process.

2. 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.

3. The method of claim 1 , wherein the method further includes clonally amplifying a portion of the at least one adapter-ligated target-specific amplicon.

4. The method of claim 3 , wherein clonally amplifying a portion of the at least one adapter-ligated target-specific amplicon includes emulsion PCR, bridge PCR or isothermal amplification.

5. The method of claim 1 , wherein the nucleic acid sample comprises cDNA or genomic DNA obtained from cell-free circulating DNA.

6. The method of claim 5 , wherein the cell-free circulating DNA is isolated from a maternal subject.

7. The method of claim 1 , wherein the adapter is a single-stranded or double stranded adapter.

8. The method of claim 7 , wherein the adapter further includes a barcode, tag or universal priming sequence.

9. The method of claim 7 , 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.

10. The method of claim 9 , wherein the ligase is an isothermal ligase.

11. The method of claim 1 , wherein the nucleic acid sample comprises cDNA or genomic DNA obtained from a sample selected from any of a fresh frozen sample, a formalin fixed, paraffin embedded (FFPE) sample, and a fine needle aspirate sample.

12. The method of claim 1 , wherein the number of different target-specific sequences amplified is in a single multiplex amplification reaction, and is about 12-plex to about 10000 different target sequences.

13. The method of claim 1 , wherein the ligation reaction includes no more than two different adapters.

14. A method of generating an amplicon library from a nucleic acid sample comprising:
a) amplifying within a single reaction mixture a multiplex of a plurality of different target sequences from a sample comprising combining a plurality of different target-specific primers 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, wherein the number of different target-specific sequences amplified during the single multiplex amplification reaction is about 12-plex to about 10000 different target sequences;
b) ligating at least one adapter to the end of the at least one of the multiplex of different target-specific amplicons and producing at least one adapter-ligated target-specific amplicon, thereby preparing an amplicon library of different adapter-ligated target sequences;

wherein each of the plurality of target specific primers have the following criteria:
(1) includes two or more modified cleavable 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; and

wherein none of the adapters in a ligation mixture, prior to ligating, can hybridize under high stringency, to any one of the target specific amplicon sequences in the reaction; and
wherein the method is optionally carried out in a single reaction, addition only process.

15. The method of claim 14 , wherein the nucleic acid sample comprises cDNA or genomic DNA.

16. The method of claim 15 , wherein the nucleic acid sample comprises DNA obtained from cell-free circulating DNA.

17. The method of claim 16 , wherein the cell-free circulating DNA is isolated from a maternal subject.

18. The method of claim 14 , wherein the adapter is a single-stranded or double stranded adapter.

19. The method of claim 18 , wherein the adapter further includes a barcode, tag or universal priming sequence.

20. The method of claim 19 , wherein each adapter further includes a universal priming sequence.

Assignees

• Life Technologies Corp

Inventors

• Leamon John
• Andersen Mark
• Thornton Michael

Classifications

• C12Q1/6886

  for cancer (immunoassay for cancer G01N33/575) · CPC title

• C12N15/1065

  Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags · CPC title

• C12Q1/686 Primary

  Polymerase chain reaction [PCR] · CPC title

• C12Q2537/143

  Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis · CPC title

• C12Q1/6855

  Ligating adaptors · CPC title