Process for nucleic acid hybridization and amplification
First Claim
1. A method of achieving synthesis and amplification of a double-stranded DNA target sequence having first and second complementary strands, each strand with 5'"'"' and 3'"'"' ends, comprising:
- (a) complexing a primer complementary to a 5'"'"' end region of the first strand and a primer complementary to a 5'"'"' end region of the second strand with heat-stable RecA protein in the presence of ATP-γ
-S;
(b) reacting the complexed primers in a reaction mixture also containing the target sequence, all four dNTPs, and a heatstable polymerase, said reacting performed above about 50°
C. and below the temperature required for thermal dissociation of the target strands and their respective primers, and said reacting continued until a desired degree of amplification of the target sequence is achieved.
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Abstract
The present invention is directed to a method of achieving RecA protein facilitated amplification of a double-stranded DNA target sequence having first and second complementary strands, each strand with 5'"'"' and 3'"'"' ends. The method involves complexing a primer complementary to a 5'"'"' end region of the first strand and a primer complementary to a 5'"'"' end region of the second strand with RecA protein in the presence of ATP-γ-S. The complexed primers are then reacted in a mixture also containing the target sequence, all four dNTPs, RecA protein and DNA polymerase. The reaction is conducted below the temperature required for thermal dissociation of the two target strands and continued until a desired degree of amplification of the target sequence is achieved.
The present invention further includes the cloning and identification of the coding sequences for the RecA protein of Thermus aquaticus.
199 Citations
4 Claims
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1. A method of achieving synthesis and amplification of a double-stranded DNA target sequence having first and second complementary strands, each strand with 5'"'"' and 3'"'"' ends, comprising:
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(a) complexing a primer complementary to a 5'"'"' end region of the first strand and a primer complementary to a 5'"'"' end region of the second strand with heat-stable RecA protein in the presence of ATP-γ
-S;(b) reacting the complexed primers in a reaction mixture also containing the target sequence, all four dNTPs, and a heatstable polymerase, said reacting performed above about 50°
C. and below the temperature required for thermal dissociation of the target strands and their respective primers, and said reacting continued until a desired degree of amplification of the target sequence is achieved. - View Dependent Claims (2, 3, 4)
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Specification
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Current AssigneeSRI International, Inc.
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Original AssigneeSRI International, Inc.
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InventorsZarling, David A., Sena, Elissa P., Green, Christopher J.
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Primary Examiner(s)YARBROUGH, AMELIA
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Application NumberUS07/520,321Time in Patent Office1,149 DaysField of Search435/6, 435/91, 435/172.3, 935/78, 935/77, 436/501, 530/825US Class Current435/91.2CPC Class CodesC12Q 1/6844 Nucleic acid amplification ...C12Q 2521/507 RecombinaseC12Q 2527/101 TemperatureC12Q 2527/125 Specific component of sampl...
