Method for hybridizing nucleic acids using single-stranded nucleic acid binding protein

• US 5,449,603 A
• Filed: 10/24/1989
• Issued: 09/12/1995
• Est. Priority Date: 10/24/1989
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California (Agilent Technologies, Inc.)
• Inventors: Mathur, Eric J., Nielson, Kirk B.
• Primary Examiner(s): BICKEL, MINDY B
• Application Number:
  US07/425,864
• Time in Patent Office: 2,149 Days
• Field of Search: 435/6, 536/27, 536/24.3, 935/77, 935/78
• US Class Current: 435/5
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins

Abstract

The present invention relates to an improved method for hybridizing polynucleotides with complementary nucleic acid sequences. Specifically, it relates to a method of increasing the specificity of a polynucleotide hybridization reaction in the presence of single-stranded nucleic acid binding protein.

Claims

1. A method for hybridizing a polynucleotide probe to a target nucleic acid, consisting essentially of:

a) forming a hybridization reaction mixture by admixing effective amounts of (i) a labeled polynucleotide probe, (ii) a target nucleic acid affixed to a solid matrix, (iii) a divalent cation, and (iv) an isolated single-stranded nucleic acid binding protein, said polynucleotide probe having a sequence substantially complementary to a predetermined sequence present on the target nucleic acid and said hybridization reaction mixture being substantially free of RecA protein; and

b) maintaining the hybridization reaction mixture under hybridizing conditions for a time period sufficient for the polynucleotide probe to hybridize to complementary nucleic acid sequences present in the target nucleic acid and form a hybridization product.

2. The method of claim 1 wherein said single-stranded nucleic acid binding protein is selected from the group consisting of Eco SSB, T4 gene 32 protein and T7 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 divalent cation is Mg⁺⁺.

5. The method of claim 1 wherein said effective amount of divalent cation is in the range of 0.5 mM to 20 mM.

6. The method of claim 1 wherein said single-stranded nucleic acid binding protein is Eco SSB and said divalent cation is Mg⁺⁺ present at a concentration of about 15 mM.

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

8. A method for detecting the presence of a specific nucleic acid sequence in a sample containing a nucleic acid, consisting essentially of:

a) forming a hybridization reaction mixture by admixing effective amounts of (i) a labeled polynucleotide probe, (ii) an unlabeled target nucleic acid, (iii) a divalent cation, and (iv) an isolated single-stranded nucleic acid binding protein, said polynucleotide probe having a sequence substantially complementary to a predetermined sequence present on the target nucleic acid and said hybridization reaction mixture being substantially free of RecA protein;

b) maintaining the hybridization reaction mixture under hybridizing conditions for a time period sufficient for the polynucleotide probe to hybridize to complementary nucleic acid sequences present in the target nucleic acid and form a hybridization product; and

c) detecting the presence of any hybridization product formed in step (b) thereby detecting the presence of the specific nucleic acid sequence in the nucleic acid.