Methods for generating single stranded DNA by the polymerase chain reaction

• US 5,066,584 A
• Filed: 09/23/1988
• Issued: 11/19/1991
• Est. Priority Date: 09/23/1988
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems (Roche Holding AG)
• Original Assignee: Cetus Corporation (Bayer AG)
• Inventors: Gyllensten, Ulf B., Erlich, Henry A.
• Primary Examiner(s): Martinell, James
• Application Number:
  US07/248,896
• Time in Patent Office: 1,152 Days
• Field of Search: 435/91, 435/6, 536/27, 436/63, 436/94, 935/17
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/143 Concentration of primer or probe
• CPC Class Codes: C12Q 2531/107 Probe or oligonucleotide ligation

Abstract

Single stranded DNA can be generated by the polymerase chain reaction using two oligonucleotide primers, one persent in a limiting concentration. The single stranded DNA is useful in procedures involving utilizing nucleic acid probes and for purposes of nucleic acid sequencing.

Claims

1. A method for generating single stranded DNA by the polymerase chain reaction, said method comprising:

(a) treating a nucleic acid with deoxyribonucleoside-5'"'"'-triphosphates, an agent for polymerization, and first and second oligonucleotide primers under hybridizing conditions such that an extension product of said first primer is synthesized that is complementary to a nucleotide sequence in said nucleic acid, wherein the extension product of said first primer can serve as a template for synthesis of an extension product of said second primer and the first primer is present in excess of the second primer;

(b) denaturing the extension products of said primers formed in step (a) from the templates on which they were synthesized;

(c) treating the products of step (b) with the primers and under the conditions of step (a); and

(d) repeating steps (b) and (c) until said second primer is present in a limiting concentration so that the extension product of the first primer accumulates in excess of the extension product of the second primer.

2. The method of claim 1, wherein said agent for polymerization is Thermus aquaticus DNA polymerase.

3. The method of claim 1, wherein said primers are present in a ratio of first primer to second primer in the range of 10:

1 to 1000;

1.

4. The method of claim 1, wherein said primers are present in a ratio of first primer:

second primer of 10;

1.

5. The method of claim 1, wherein said primers are present in a ratio of first primer:

second primer of 50;

1.

6. The method of claim 1, wherein said primers are present in a ratio of first primer:

second primer of 100;

1.

7. The method of claim 1, wherein said primers are present in a ratio of first primer:

second primer of 1000;

1.