Arbitrarily primed polymerase chain reaction method for fingerprinting genomes

• US 5,487,985 A
• Filed: 10/09/1992
• Issued: 01/30/1996
• Est. Priority Date: 10/15/1990
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Stratagene California
• Inventors: Sorge, Joseph A., McClelland, Michael, Welsh, John T.
• Primary Examiner(s): Pahr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/959,119
• Time in Patent Office: 1,208 Days
• Field of Search: 435/91, 435/6, 435/91.1, 435/91.2, 935/77, 935/78, 935/88
• 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 rapid method for generating a set of discrete DNA amplification products characteristic of a genome as a "fingerprint" comprises the steps of: priming target nucleic acid of a genome or from a cellular RNA preparation with an single-stranded primer to form primed nucleic acid such that a substantial degree of internal-mismatching occurs between the primer end the target nucleic acid; amplifying the primed nucleic acid by performing at least one cycle of polymerase chain reaction amplification; and amplifying the product of step (2) by performing at least about 10 cycles of polymerase chain reaction amplification. The method is known as the arbitrarily primed polymerase chain reaction (AP-PCR) method and is suitable for the identification of bacterial species and strains, including Staphylococcus and Streptococcus species, mammals and plants. The method of the present invention can identify species, cell types or tissues rapidly, using only a small amount of biological material, and does not require knowledge of the nucleotide sequence or other molecular biology of the nucleic acids of the organisms to be identified. The method can also be used to generate detectable polymorphisms for use in genetic mapping of animals and humans, and be used to detect differential gene expression within tissues

Claims

1. A method of generating a set of discrete DNA segments characteristic of a genome comprising:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA and at least one first polynucleotide primer from about 10 to about 50 nucleotide bases in length;

(b) subjecting said PCR admixture of step (a) to at least one PCR thermocycle, each of said thermocycles comprising hybridization, primer extension and denaturation phases, said hybridization phase comprising hybridization conditions permitting the arbitrary priming of said genomic DNA, thereby producing said set of discrete DNA segments;

(c) contacting, in a PCR buffer, the set of discrete DNA amplification products formed in step (b) with at least one second polynucleotide primer from about 10 to 50 nucleotides in length, wherein said second primer or primers of this step each have nucleotide sequences that match the first primer or primers used in step (a) except that the second primer(s) each have one or more additional nucleotide bases at the 3'"'"' terminus of each second primer, wherein the additional nucleotide bases are additional with respect to the 3'"'"' terminus of the first primer or primers, to form a second PCR admixture; and

(d) 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 a subset of said discrete DNA segments.

2. The method of claim 1 further comprising the steps of:

(e) applying the amplified subset of discrete DNA segments produced in step (d) to a separating apparatus; and

(f) size-separating the applied segments into bands within the separating apparatus to form a fingerprint of segments characteristic of said genome.

3. The method of claim 1 wherein said second primer or primers of step (c) have from one to four additional nucleotide bases at said 3'"'"' termini.

4. The method of claim 1 wherein the first primer of step (a) is at least about 17 bases in length and less than about 40 bases in length.

5. The method of claim 1 wherein said hybridization conditions in step (b) comprise a hybridization temperature in the range of about 35°

C. to about 55°

C.

6. The method of claim 5 wherein said hybridization temperature is about 40°

C. to about 48°

C.

7. The method of claim 1 wherein said hybridization conditions in step (d) comprise a hybridization temperature greater than about 55°

C.

8. The method of claim 7 wherein said hybridization temperature is about 60°

C.

9. The method of claim 1 wherein said hybridization conditions in step (b) comprise a hybridization temperature of about 40°

C. to about 50°

C., and said hybridization conditions in step (d) comprise a hybridization temperature of about 60°

C.

10. The method of claim 1 wherein said plurality of PCR thermocycles of step (d) is about 10 to about 40 PCR thermocycles.

11. A method for generating a discrete set of DNA segments characteristic of a sample of 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;

(d) subjecting said PCR admixture of step (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 amplification products formed in step (d) with at least one third polynucleotide primer from about 10 to 50 nucleotides in length, wherein said third primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (c) except that the third primer(s) each have one or more additional nucleotide bases at the 3'"'"' terminus of each primer, wherein the additional nucleotide bases are additional with respect to the 3'"'"' terminus of the second primer or primers, 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 mismatch, thereby amplifying a subset of said discrete DNA segments.

12. The method of claim 11 wherein said third primer or primers of step (e) have from one to four additional nucleotide bases at said 3'"'"' termini.

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

(a) forming a polymerase chain reaction (PCR) admixture by combining said RNA sample, at least one polynucleotide primer from about 10 to about 50 nucleotide bases in length, and a PCR buffer that contains 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 step (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 amplification products formed in step (c) with at least one polynucleotide primer from about 10 to 50 nucleotides in length, wherein said primer or primers of this step each have nucleotide sequences that match the primer or primers used in step (a) except that the primer(s) step each have one or more additional nucleotide bases at the 3'"'"' terminus of each primer to form a second PCR admixture; and

(e) subjecting said second PCR admixture to 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 mismatch, thereby amplifying a subset of said discrete DNA segments.

14. The method of claim 13 wherein said primer of primers of step (e) have from one to four additional nucleotide bases at said 3'"'"' termini.