Methods and compositions for use in high fidelity polymerase chain reaction

• US 5,976,842 A
• Filed: 10/30/1997
• Issued: 11/02/1999
• Est. Priority Date: 10/30/1997
• Status: Expired due to Term

• Current Assignee: Takara Bio Usa, Inc. (Ningbo Junsheng Investment Group Co., Ltd.)
• Original Assignee: Clontech Laboratories Incorporated (Ningbo Junsheng Investment Group Co., Ltd.)
• Inventors: Wurst, Helmut
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): Siew, Jeffrey
• Application Number:
  US08/960,718
• Time in Patent Office: 733 Days
• Field of Search: 435/91.1, 435/91.2, 435/188, 435/194, 435/6, 536/23.1
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

Methods and compositions are provided for performing high fidelity polymerase chain reactions. In the subject methods, primer extension products are produced with a low error frequency rate through the use of at least one of unequal concentrations of dNTPs or a melting point reducing agent. Also provided are kits and reagent mixtures for use in the subject methods. The subject invention finds use in a variety of applications, particularly in applications where high fidelity PCR is desired.

Claims

1. A method of performing a high fidelity polymerase chain reaction (PCR), said method comprising:

(a) contacting a template DNA nucleic acid with a PCR reagent mixture that comprises both a Family A polymerase and a Family B polymerase and provides for an error frequency rate that does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle; and

(b) performing a plurality of reaction cycles, wherein each cycle comprises;

(i) denaturation;

(ii) annealing; and

(iii) polymerization;

whereby a polymerase chain reaction is performed in which the error frequency rate does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle.

2. The method according to claim 1, wherein said reagent mixture further comprises unequal concentrations of dNTPs.

3. The method according to claim 1, wherein said reagent mixture further comprises a melting point reducing agent.

4. A method of performing a high fidelity polymerase chain reaction (PCR), said method comprising:

(a) contacting a template nucleic acid with primer and a reagent mixture comprising at least one Family A and Family B polymerase and at least one of;

unequal amounts of dNTPs and a melting point reducing agent, wherein said reagent mixture provides for an error frequency rate that does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle; and

(b) performing a plurality of reaction cycles, wherein each cycle comprises;

(i) a denaturation;

(ii) annealing; and

(iii) polymerization

whereby a polymerase chain reaction is performed in which the error frequency rate does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle.

5. The method according to claim 4, wherein said reagent mixture comprises unequal concentrations of dNTPs.

6. The method according to claim 4, wherein said reagent mixture comprises a melting point reducing agent.

7. The method according to claim 4, wherein said reagent mixture is further characterized by the presence of more Family A polymerase than Family B polymerase.

8. A method of performing a high fidelity polymerase chain reaction (PCR), said method comprising:

(a) contacting a template DNA with primer and a reagent mixture that provides for an error frequency rate that does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle, wherein said reagent mixture comprises;

(i) at least one Family A and Family B polymerase;

(ii) unequal concentrations of dNTPs; and

(iii) urea; and

(b) performing a plurality of reaction cycles, wherein each cycle comprises;

(i) denaturation;

(ii) annealing; and

(iii) polymerization;

whereby a polymerase chain reaction is performed in which the error frequency rate does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle.

9. The method according to claim 8, wherein said reagent mixture comprises more Family A polymerase than Family B polymerase.

10. The method according to claim 9, wherein said Family A polymerase is selected from the group consisting of a Taq polymerase, a Tth polymerase, and homologues thereof.

11. The method according to claim 9, wherein said Family B polymerase is selected from the group consisting of a Vent polymerase, a Deep Vent polymerase, a Pfu polymerase and a Pwo polymerase.

12. The method according to claim 8, wherein said unequal concentrations of dNTPs are characterized by at least one of a greater amount of dATP and a lesser amount of dGTP with respect to the remaining dNTPs.

13. The method according to claim 8, wherein said urea is present in an amount ranging from about 20 to 500 mM.

14. The method according to claim 8, wherein said plurality of cycles comprises from about 5 to 40 cycles.

15. A method of performing a high fidelity polymerase chain reaction (PCR), said method comprising:

(a) contacting a template DNA with primer and reagents to produce a reaction mixture that provides for an error frequency rate that does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle, wherein said reagents comprise;

(i) a Family A polymerase and Deep Vent polymerase, wherein said Family A polymerase is present in an amount greater than said Deep Vent polymerase; and

(ii) unequal concentrations of dNTPs, wherein said unequal concentrations of dNTPs are characterized by at least one of a greater amount of dATP and a lesser amount of dGTP with respect to the remaining dNTPs; and

(b) performing from about 5 to 40 reaction cycles, wherein each cycle comprises;

(i) denaturation;

(ii) annealing; and

(iii) polymerization;

whereby a polymerase chain reaction is performed in which the error frequency rate does not exceed 1.3×

10^-6 mutations per base pair per PCR cycle.

16. The method according to claim 15 wherein the pH of said reaction mixture ranges from about 6.0 to 9.5

17. The method according to claim 15, wherein said reaction mixture is characterized by having a magnesium ion concentration ranging from about 0.5 to 10 mM.

18. A reagent mixture for use in high fidelity polymerase chain reactions, said mixture comprising

(a) at least one Family A polymerase;

(b) at least one Family B polymerase; and

(c) urea.

19. The reagent mixture according to claim 18, wherein said reagent mixture further comprises unequal concentration of dNTPs.

20. The reagent mixture according to claim 19, wherein said urea is present in an amount ranging from about 20 to 500 mM.

21. The reagent mixture according to claim 18, wherein said Family A polymerase is selected from the group consisting of a Taq polymerase and a Tth polymerase.

22. The reagent mixture according to claim 18, wherein said Family B polymerase is selected from the group consisting of a Vent polymerase, a Deep Vent polymerase, a Pfu polymerase and a Pwo polymerase.

23. A reagent mixture for use in high fidelity polymerase chain reactions, said mixture comprising:

(a) a Family A polymerase and Deep Vent polymerase, wherein said Family A polymerase is present in amount greater than said Deep Vent polymerase;

(b) unequal concentrations of dNTPS, wherein said unequal concentrations of dNTPs are characterized by at least one of a greater amount of dATP and a lesser amount of dGTP with respect to the remaining dNTPs.

(c) urea in an amount ranging from about 20 to 500 mM.

24. A kit for use in performing high fidelity polymerase chain reactions, said kit comprising:

(a) at least one Family A polymerase;

(b) at least one Family B polymerase; and

(c) urea.

25. The kit according to claim 24, wherein said Family A polymerase is selected from the group consisting of a Taq polymerase and a Tth polymerase.

26. The kit according to claim 24, wherein said Family B polymerase is selected from the group consisting of a Vent polymerase, a Deep Vent polymerase, a Pfu polymerase and a Pwo polymerase.

27. The kit according to claim 24, wherein said kit further comprises unequal concentrations of dNTPs.

28. The kit according to claim 24, wherein said urea is present in an amount ranging from about 20 to 500 mM.

29. A kit for use in high fidelity polymerase chain reactions, said kit comprising:

(a) a Family A polymerase and Deep Vent polymerase, wherein said Family A polymerase is present in amount greater than said Deep Vent polymerase;

(b) unequal concentrations of dNTPs, wherein said unequal concentrations of dNTPs are characterized by at least one of a greater amount of dATP and a lesser amount of dGTP with respect to the remaining dNTPs; and

(c) urea in an amount ranging from about 20 to 500 mM.