Method for producing primed nucleic acid templates

• US 5,773,257 A
• Filed: 06/06/1995
• Issued: 06/30/1998
• Est. Priority Date: 10/24/1989
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Nielson, Kirk B., Mathur, Eric J.
• Primary Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US08/469,564
• Time in Patent Office: 1,120 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 530/350, 935/77, 935/78
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• 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 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label

Abstract

The present invention relates to an improved method for producing primed nucleic acid templates. Specifically, it relates to a method, compositions and kits therefor, of increasing the specificity of primer extension reactions by hybridizing primer to template in the presence of single-stranded nucleic acid binding protein.

Claims

1. A method of preparing a strand of nucleic acid having a nucleotide sequence substantially complementary to a template nucleic acid, which method comprises:

(a) forming a primer extension reaction admixture whereby nonspecific hybridization of polynucleotide synthesis primer is reduced by admixing said template nucleic acid with a hybridization effective amount of (i) said polynucleotide synthesis primer having a nucleotide sequence complementary to a portion of the template nucleic acid, (ii) an isolated single-stranded nucleic acid binding protein (SSB), (iii) a divalent cation, and (iv) an enzyme capable of inducing polynucleotide synthesis in the presence of a primed template; and

(b) maintaining the primer extension reaction admixture under polynucleotide synthesizing conditions for a time period sufficient for said enzyme to produce a primer extension product, thereby producing said strand of nucleic acid.

2. The method of claim 1 wherein said single-stranded nucleic acid binding protein is Eco SSB.

3. The method of claim 1 wherein said divalent cation is selected from the group consisting of Mg⁺⁺, Mn⁺⁺, Ca⁺⁺ and Zn⁺⁺.

4. The method of claim 1 wherein said hybridization effective amount of single-stranded nucleic acid binding protein is in the range of 1 ng to 10 ug per 100 ng of template nucleic acid, and said divalent cation is Mg⁺⁺ present at a concentration of 1.5 mM.

5. A method of preparing a strand of DNA having a nucleotide sequence substantially complementary to a template nucleic acid, which method comprises:

(a) forming a primer extension reaction admixture whereby nonspecific hybridization of polynucleotide synthesis is reduced by admixing said template nucleic acid with a hybridization effective amount of (i) a polynucleotide synthesis primer having a nucleotide sequence complementary to a portion of the template nucleic acid, (ii) isolated Eco SSB, (iii) Mg⁺⁺, and (iv) a DNA polymerase; and

(b) maintaining the primer extension reaction admixture under polynucleotide synthesizing conditions for a time period sufficient for said DNA polymerase to produce a primer extension product, thereby producing said strand of DNA.

6. A method of amplifying a specific nucleic acid sequence present in a sample containing a double-stranded nucleic acid molecule, which method comprises:

(a) separating the strands of the double-stranded nucleic acid molecule to form single-stranded templates;

(b) treating the single-stranded templates with hybridization effective amounts of (i) primers that are selected so as to be complementary to portions of the different strands of the specific double-stranded sequence to hybridize therewith such that an extension product synthesized from one primer, when it is separated from its template nucleic acid strand, can serve as a template for synthesis of the extension product of the other primer, (ii) an isolated single-stranded nucleic acid binding protein (SSB) whereby nonspecific hybridization of said primers is reduced, (iii) a divalent cation, and (iv) an enzyme capable of producing primer extension products from said single-stranded templates, wherein said treatment is conducted under conditions such that the primers hybridize to the specific sequences and an extension product of each primer is synthesized by the action of the enzyme, said product being substantially complementary to each single-stranded template;

(c) separating the primer extension products formed in step (b) from the templates on which they are synthesized to produce single-stranded molecules; and

(d) treating the single-stranded molecules produced in step (c) with hybridization effective amounts of the primers, SSB, divalent cation and enzyme of step (b) under conditions such that a primer extension product is synthesized using the single-stranded molecules formed in step (c) as a template, thereby amplifying said specific nucleic acid sequence.

7. The method of claim 6 wherein said single-stranded nucleic acid binding protein is selected from the group consisting of Eco SSB, T4 gene 32 protein and T7 SSB.

8. The method of claim 6 wherein said enzyme is a DNA polymerase selected from the group consisting of Klenow fragment, Taq DNA polymerase, recombinant Taq DNA polymerase, T7 DNA polymerase, modified T7 DNA polymerase and T4 DNA polymerase.

9. The method of claim 6 wherein said divalent cation is selected from the group consisting of Mg⁺⁺, Mn⁺⁺, Ca⁺⁺ and Zn⁺⁺.

10. The method of claim 9 wherein said hybridization effective amount of divalent cation is in the range of 0.5 mM to 20 mM.

11. The method of claim 9 wherein said divalent cation is Mg⁺⁺ present at a concentration of about 1.5 mM.

12. The method of claim 6 wherein said hybridization effective amount of single-stranded nucleic acid binding protein is in the range of 1 ng to 10 ug per 100 ng of template nucleic acid.

13. The method of claim 6 wherein said separating in steps (a) and (d) is accomplished by heat denaturing the double-stranded nucleic acid molecule.

14. The method of claim 13 wherein said single-stranded nucleic acid binding protein is heat stable.

15. The method claim 14 wherein said single-stranded nucleic acid binding protein is Eco SSB.

16. The method of claim 13 wherein said DNA polymerase is a heat-stable DNA polymerase.

17. The method of claim 16 wherein said heat-stable DNA polymerase is Taq DNA polymerase or recombinant Taq DNA polymerase.