Polymerase compositions and kits, and methods of using and making the same

Patent metadata

Field	Value
Publication number	US-10344268-B2
Application number	US-201615277834-A
Country	US
Kind code	B2
Filing date	Sep 27, 2016
Priority date	Oct 1, 2015
Publication date	Jul 9, 2019
Grant date	Jul 9, 2019

Abstract

The present disclosure provides compositions, methods, kits, systems and apparatus that are useful for nucleic acid polymerization. In particular, recombinant polymerases and biologically active fragments thereof are provided that allow for nucleic acid amplification. In some aspects, the disclosure provides recombinant polymerases that yield lower systematic error rates and/or improved accuracy, when used in sequencing by synthesis reactions as compared to a control polymerase. In one aspect, the disclosure relates to recombinant polymerases useful for nucleic acid sequencing, genotyping, copy number variation analysis, paired-end sequencing and other forms of genetic analysis. In another aspect, the recombinant polymerases are useful for the amplification of nucleic acid templates during PCR, emPCR, isothermal amplification, recombinase polymerase amplification, rolling circle amplification, strand displacement amplification and proximity ligation amplification. In some aspects, the disclosure relates to recombinant polymerases useful for the generation of nucleic acid libraries and/or nucleic acid templates.

First claim

What is claimed is:

1. A composition comprising a recombinant DNA polymerase, wherein the recombinant DNA polymerase comprises a polypeptide segment exhibiting polymerase activity, wherein said polypeptide segment consists of the amino acid sequence of SEQ ID NO: 35, except that said polypeptide segment has
a) a single amino acid substitution selected from the group consisting of E30K, G209Q, E220H, E220T, P236I, P236R, E245R, V251R, L252R, S260K, D264M, E267M, E267K, E267R, E277T, T324R, A344Y, E442R, E442Y, E456R, D480L, A492K, E493G, M495C, N517C, N517K, and E522C,
b) amino acid substitutions L252R and H528T, or
c) amino acid substitutions L252R, H528T, and H473G,
wherein the numbering of said amino acid substitutions in the polypeptide segment is relative to SEQ ID NO: 35; and
wherein the recombinant DNA polymerase yields a lower error rate, increased signal, and/or improved accuracy when used in a sequencing by synthesis reaction where hydrogen ions are detected, as compared to a reference polymerase having the amino acid sequence of SEQ ID NO: 35.

2. The composition of claim 1 , wherein the polypeptide segment has the amino acid substitutions L252R and H528T.

3. The composition of claim 2 , wherein the polypeptide segment further has amino acid substitution H473G.

4. The composition of claim 3 , wherein the recombinant DNA polymerase consists of the sequence of SEQ ID NO: 119.

5. The composition of claim 1 , wherein at least 90% of the amino acid sequence of the recombinant DNA polymerase is from the polypeptide segment.

6. The composition of claim 5 , wherein the polypeptide segment has the amino acid substitutions L252R and H528T.

7. The composition of claim 6 , wherein the polypeptide segment further has amino acid substitution H473G.

8. A method for performing a polymerization reaction, comprising
(a) contacting a recombinant DNA polymerase according to claim 1 with a nucleic acid template in the presence of one or more nucleotides; and
(b) polymerizing at least one of the one or more nucleotides using the recombinant polymerase.

9. The method of claim 8 , wherein the DNA polymerase is contacted with the nucleic acid template in the presence of one or more nucleotides and a salt, wherein the salt is KCl and/or NaCl, and wherein the salt concentration is from 125 mM to 175 mM.

10. A method for obtaining sequence information from a nucleic acid template, comprising:
(a) providing a reaction mixture comprising a template nucleic acid, one or more nucleotides, a sequencing primer and a recombinant polymerase according to claim 1 ;
(b) contacting the template nucleic acid with at least one type of nucleotide, wherein the contacting includes incorporating one or more nucleotides from the at least one type of nucleotide onto the 3′ end of the sequencing primer and generating an extended primer product; and
(c) detecting the presence of the extended primer product in the reaction mixture by detecting a release of hydrogen ions, thereby determining whether nucleotide incorporation has occurred.

11. The method of claim 10 , wherein the DNA polymerase is contacted with the nucleic acid template in the presence of one or more nucleotides and a salt, wherein the salt is KCl and/or NaCl, and wherein the salt is present at a concentration of between 125 mM and 175 mM.

12. The method of claim 10 , wherein the recombinant DNA polymerase has the sequence of SEQ ID NO: 119.

13. The composition according to claim 1 , wherein said recombinant DNA polymerase comprises the 295 amino terminal amino acids of SEQ ID NO: 120 followed by the polypeptide segment.

14. The composition of claim 13 , wherein the polypeptide segment has the amino acid substitutions L252R and H528T.

15. The composition of claim 14 , wherein the polypeptide segment further has amino acid substitution H473G.

16. The composition of claim 13 , wherein at least 90% of the amino acids of the recombinant DNA polymerase are from the 295 amino terminal amino acids of SEQ ID NO: 120 and the polypeptide segment.

17. The composition of claim 16 , wherein the polypeptide segment has the amino acid substitutions L252R and H528T.

18. The composition of claim 17 , wherein the polypeptide segment further has amino acid substitution H473G.

19. A composition comprising a recombinant DNA polymerase, wherein the recombinant DNA polymerase comprises a polypeptide segment exhibiting polymerase activity, wherein said polypeptide segment consists of the amino acid sequence of SEQ ID NO: 121.

20. A method for performing a polymerization reaction, comprising:
(a) contacting a recombinant DNA polymerase according to claim 19 with a nucleic acid template in the presence of one or more nucleotides; and
(b) polymerizing at least one of the one or more nucleotides using the recombinant polymerase.

21. A method for obtaining sequence information from a nucleic acid template, comprising:
(a) providing a reaction mixture comprising a template nucleic acid, one or more nucleotides, a sequencing primer and a recombinant polymerase according to claim 19 ;
(b) contacting the template nucleic acid with at least one type of nucleotide, wherein the contacting includes incorporating one or more nucleotides from the at least one type of nucleotide onto the 3′ end of the sequencing primer and generating an extended primer product; and
(c) detecting the presence of the extended primer product in the reaction mixture by detecting a release of hydrogen ions, thereby determining whether nucleotide incorporation has occurred.

Assignees

• Life Technologies Corp

Inventors

• Mazur Daniel
• Vander Horn Peter
• Tozer Eileen
• Chen Sihong
• Luo Guobin
• Shirley Joshua
• HEINEMANN Kevin

Classifications

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q2521/101

  DNA polymerase · CPC title

• C12Q1/6844

  Nucleic acid amplification reactions · CPC title