Polymerase chain reaction amplification method using a single primer which randomly anneals

• US 5,665,572 A
• Filed: 08/23/1994
• Issued: 09/09/1997
• Est. Priority Date: 08/30/1991
• Status: Expired due to Fees

• Current Assignee: Research Development Corporation Of Japan (Japan Science And Technology Agency)
• Original Assignee: Research Development Corporation Of Japan (Japan Science And Technology Agency)
• Inventors: Ikeda, Joh-E, Yokoi, Haruhiko, Hadano, Shinji
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US08/294,606
• Time in Patent Office: 1,113 Days
• Field of Search: 435/6, 435/91.2, 536/24.3, 536/24.31, 536/24.32, 536/24.33
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2527/113 Time
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/153 Viscosity

Abstract

A method of amplifying template DNA by polymerase chain reaction (PCR) in which a single oligonucleotide primer having a restriction site is contacted with the template DNA, whereby the oligonucleotide randomly anneals to a single strand of the template DNA and DNA sequences complementary to the single strand are synthesized. An initial PCR amplification yields synthetic DNA sequences having the oligonucleotide sequence incorporated therein at the 5'"'"' end, and a sequence complementary to the template DNA. A second PCR amplification under higher stringency conditions amplifies regions of the template DNA to give DNA fragments having the restriction sites of the oligonucleotide primer. Thereby the method can be used to amplify trace quantities of template DNA of unknown sequence simply and efficiently, which has applications in the construction of DNA libraries of chromosome specific regions and the development of probes for chromosome mapping.

Claims

1. A method for amplifying template DNA having an unknown sequence by a polymerase chain reaction (PCR) using oligonucleotide primers comprising a contiguous sequence which consists of SEQ ID No. 1, which method comprises the steps of:

(a) conducting multiple PCR cycles on a first reaction solution containing said template DNA, said oligonucleotide primers, and an amphipathic polymer, each PCR cycle comprising;

(i) denaturing double-stranded DNA into single-stranded DNA, wherein in the first of said multiple PCR cycles the double-stranded DNA is said template DNA,

(ii) annealing said oligonucleotide primers to the single-stranded DNA at 10°

-40°

C. for 90-150 minutes, and

(iii) synthesizing extension products of said oligonucleotide primers, which products are complementary duplicates of Various regions of the single-stranded DNA,

wherein the amphipathic polymer increases the annealing efficiency of the oligonucleotide primers and single-stranded DNA,

so that a plurality of extended primer molecules are synthesized each having the nucleotide sequence of said oligonucleotide primer at both ends and a nucleotide sequence of each region of the template DNA; and

(b) conducting multiple PCR cycles under stringency conditions higher than in step (a) on a second reaction solution containing the reaction solution produced by step (a) and an additional amount of said oligonucleotide primers, but not containing said amphipathic polymer, thereby amplifying the template DNA as a group of various DNA sequences therein.

2. The method of claim 1, wherein the amphipathic polymer is a polyethylene glycol having a molecular weight in a range of 1,000 to 10,000.

3. The method of claim 1, wherein step (a) the amphipathic polymer is polyethylene glycol present in the range of 10 to 15% and the oligonucleotide primers are present in the range of 1.0 to 1.5 μ

M in the first reaction solution.

4. The method of claim 1, wherein in step (b) the additional oligonucleotide primers are present in the range of 4.0 to 5.0 μ

M in the second reaction solution.

5. A kit for amplifying a template DNA having an unknown sequence by the method of claim 1 comprising in combination:

(a) oligonucleotide primers comprising a contiguous sequence which consists of SEQ ID No. 1;

(b) Tag DNA polymerase; and

(c) nucleotide triphosphates.