Methods and compositions for multiplex PCR

Patent metadata

Field	Value
Publication number	US-9885076-B2
Application number	US-201514796696-A
Country	US
Kind code	B2
Filing date	Jul 10, 2015
Priority date	Apr 28, 2011
Publication date	Feb 6, 2018
Grant date	Feb 6, 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 preparing a library of different target nucleic acid molecules from a sample comprising:
a) amplifying within a single amplification reaction mixture a multiplex of different target nucleic acid molecules from a sample with a pool of a plurality of target-specific primers, and a polymerase under amplification conditions, thereby producing a plurality of different target-specific molecules, and treating to form blunt-ended amplified target specific molecules; and
b) ligating via blunt end ligation at least one adapter to at least one of the plurality of different amplified target-specific molecules, thereby
producing a library of adapter-ligated amplified target sequences,
wherein the method includes using no more than two target-specific primers to produce any one of the adapter-ligated amplified target sequences
wherein the number of different target-specific sequences amplified during the single multiplex amplification reaction is at least 10 to about 10000 different target sequences,
wherein none of the adapters in the ligation reaction hybridizes under high stringency conditions to any one of the multiplex of different amplified target specific sequences, and
wherein each of the plurality of target specific primers has any one or more of 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; and
wherein the steps of preparing the library are carried out in a single reaction vessel comprising addition only steps.

2. The method of claim 1 , wherein each of the plurality of target specific primers comprises the two or more modified nucleotides selected from a cleavable group of 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 plurality of different target nucleic acid molecules are RNA.

4. The method of claim 3 , wherein the method further includes reverse transcribing the RNA into a population of cDNA molecules prior to the amplifying.

5. The method of claim 1 , wherein the plurality of different target nucleic acid molecules are obtained from genomic DNA in an amount from 1 ng to 10 ng of total genomic DNA.

6. A method of amplifying a plurality of different target nucleic acid sequences comprising:
a) contacting a portion of the plurality of different target sequences with a plurality of target-specific primers and a polymerase under amplification conditions,
b) producing a plurality of different amplified target sequences by extending one or more of the plurality of target-specific primers, wherein a plurality of the target-specific primers include a cleavable group and at least one of the plurality of the amplified target sequences includes a primer-derived sequence containing the cleavable group;
c) cleaving the cleavable group of the primer-derived sequence of the at least one amplified target sequence,
d) forming a cleaved end of the at least one amplified target sequence; and
e) ligating at least one adapter to the cleaved end of the at least one amplified target sequence, wherein the at least one adapter is not complementary to 20 contiguous nucleotides from a 3′ end or a 5′ end of the at least one amplified target sequence;
wherein each of the plurality of target specific primers has any one or more of 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; and
wherein the steps are carried out in a single reaction vessel comprising addition only steps.

7. The method of claim 6 , wherein the method includes using no more than two target-specific primers from the plurality of target-specific primers to produce any one of the plurality of different amplified target sequences.

8. The method of claim 6 , wherein the plurality of different target nucleic acid sequences are obtained from 1 ng to 10 ng of total genomic DNA.

9. The method of claim 1 , wherein the plurality of different target nucleic acid molecules are genomic DNA obtained from cell-free circulating DNA.

10. The method of claim 9 , wherein the cell-free circulating DNA is isolated from a maternal subject.

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

12. The method of claim 11 , wherein the adapter further includes a barcode, tag or universal priming sequence.

13. The method of claim 11 , wherein the adapter is a double stranded adapter ligated to the end of at least one digested different target specific amplicon using a ligase capable of blunt-ended ligation.

14. The method of claim 13 , wherein the ligase is an isothermal ligase.

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

16. The method of claim 15 , wherein clonally amplifying a portion of the at least one adapter-ligated target-specific amplicon includes emulsion PCR, bridge PCR or isothermal amplification.

Assignees

• Life Technologies Corp

Inventors

• Leamon John
• Andersen Mark
• Thornton Michael

Classifications

• C12N15/1065

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

• C12Q1/6874

  involving nucleic acid arrays, e.g. sequencing by hybridisation · CPC title

• C12Q2525/191

  incorporating an adaptor · 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

• C12Q2521/307

  Single strand endonuclease · CPC title