Method of primer-extension preamplification PCR

• US 6,365,375 B1
• Filed: 03/16/1999
• Issued: 04/02/2002
• Est. Priority Date: 03/26/1998
• Status: Expired due to Term

• Current Assignee: Roche Diagnostics GmbH (Roche Holding AG)
• Original Assignee: Josef Ruschoff, Wolfgang Dietmaier
• Inventors: Dietmaier, Wolfgang, Ruschoff, Josef
• Primary Examiner(s): MAUPIN, CHRISTINE L
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/270,933
• Time in Patent Office: 1,113 Days
• Field of Search: 435/91.2, 435/91.1, 435/6
• US Class Current: 435/91.1
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 2521/537 Protease
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences

Abstract

The invention provides a method for the amplification of nucleic acid fragments from a sample that comprises two or three thermocyclic amplification reactions, whereby completely randomized primers are used in the first amplification reaction and specific primers are used in the second amplification reaction, characterized in that, to amplify the DNA, a mixture of at least two DNA polymerases is used, at least one of which possesses proofreading activity. Using this method, DNA from single cells or cell clones with a low cell count can be used in mutation analysis.

Claims

1. A method for the amplification of nucleic acid fragments from a sample of cells, said method comprising treating said sample of cells with a protease, followed by two thermocyclic amplification reactions, wherein first amplification reaction is carried out using completely randomized primers, second amplification reaction is carried out using specific primers, and said first and second amplification reactions are carried out using the same mixture of at least two DNA polymerases, at least one of which possesses 3′

-5′

exonuclease activity.

2. The method of claim 1, wherein, in said first amplification reaction, the temperature at which primer extension is carried out is increased in at least some of the successive amplification cycles.

3. The method of claim 1, wherein said mixture of DNA polymerases comprises a DNA polymerase without 3′

-5′

exonuclease activity and a DNA polymerase with 3′

-5′

exonuclease activity.

4. The method of claim 1, wherein said protease is proteinase K.

5. The method of claim 3, wherein said mixture of DNA polymerases comprises Taq DNA polymerase and Pwo DNA polymerase.