Arbitrarily primed polymerase chain reaction method for fingerprinting genomes

• US 6,696,277 B1
• Filed: 03/01/1995
• Issued: 02/24/2004
• Est. Priority Date: 10/15/1990
• Status: Expired due to Fees

• Current Assignee: Stratagene California
• Original Assignee: California Institute Of Bio. Research, Stratagene California
• Inventors: McClelland, Michael, Welsh, John T., Sorge, Joseph A.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/397,335
• Time in Patent Office: 3,282 Days
• Field of Search: 435/6, 435/810, 435/91.1, 435/91.2, 436/501, 536/23.1, 536/24.1, 536/24-- 2, 536/25.3, 935/77, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A method for generating a discrete set of DNA segments characteristic of a sample of a single-stranded RNA is provided.

Claims

1. A method for generating a discrete set of DNA segments characteristic of a sample of a single-stranded RNA, which method comprises:

(a) forming a primer extension reaction admixture by combining, in a primer extension buffer, said RNA sample and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length;

(b) maintaining said primer extension reaction under primer extension conditions to produce a hybrid DNA-RNA molecule;

(c) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, said DNA-RNA hybrid molecule and at least one second polynucleotide primer from about 10 to about 50 nucleotide bases in length, wherein the first and second primer may be the same or different from one another; and

(d) subjecting said PCR admixture of (c) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension, and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments;

(e) contacting, in a PCR buffer, the set of discrete DNA segments formed in (d) with the second primer or primers used in (c) to form a second PCR admixture; and

(f) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments.

2. The method of claim 1, wherein the at least one PCR thermocycle in (d) comprises a plurality of PCR thermocycles.

3. A method for generating a discrete set of DNA segments characteristic of a sample of a single-stranded RNA, which method comprises:

(a) forming a polymerase chain reaction (PCR) admixture by combining said RNA sample, at least one polynucleotide from about 10 to about 50 nucleotide bases in length, and a PCR buffer that comprises both reverse transcriptase and thermostable DNA polymerase;

(b) maintaining said PCR admixture under reverse transcriptase primer extension conditions to produce a hybrid DNA-RNA molecule;

(c) subjecting said PCR admixture of (b) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension, and denaturation phases, said hybridization phase comprising hybridization conditions compatible with arbitrary priming of said DNA-RNA hybrid molecule, thereby producing said set of discrete DNA segments;

(d) contacting, in a PCR buffer, the set of discrete DNA segments formed in (c) with the primer or primers used in (a) to form a second PCR admixture; and

(e) subjecting said second PCR admixture to a plurality of PCR thermocycles, each of said thermocycles including hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions that do not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments.