Arbitrarily primed polymerase chain reaction method for fingerprinting genomes

• US 5,861,245 A
• Filed: 06/06/1995
• Issued: 01/19/1999
• Est. Priority Date: 10/15/1990
• Status: Expired due to Fees

• Current Assignee: Stratagene California
• Original Assignee: Stratagene and California Institute of Biological Research
• Inventors: Welsh, John Thomas, Sorge, Joseph A., McClelland, Michael
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Whisenant, Ethan
• Application Number:
  US08/471,994
• Time in Patent Office: 1,323 Days
• Field of Search: 435/6, 435/91.2, 435/91.1, 935/77, 935/78
• US Class Current: 435/6.12
• 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 with an single-stranded primer to form primed nucleic acid such that a substantial degree of internal-mismatching occurs between the primer and 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 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. Only one primer sequence is required for amplification and/or identification. The method can also be used to generate detectable polymorphisms for use in genetic mapping of animals and humans.

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 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 a low stringency hybridization temperature permitting the arbitrary priming of said genomic DNA, thereby producing said set of discrete DNA segments;

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

(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 a high stringency hybridization temperature which does 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 further comprising the step of:

(e) size-separating said amplified set of discrete DNA segments into bands to form a fingerprint of segments characteristic of said genome.

3. The method of claim 1 wherein said at least one polynucleotide primer comprises at least two different polynucleotides.

4. The method of claim 1 herein said at least one polynucleotide primer is selected from the group consisting of:

T-T-G-T-A-A-A-A-C-G-A-G-G-C-C-A-G-T;

G-G-A-A-A-C-A-G-C-T-A-T-G-A-C-C-A-T-G-A;

G-T-A-A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G;

G-C-A-A-T-T-A-A-C-C-C-T-C-A-C-T-A-A-A-G;

C-C-A-G-C-T-C-G-A-C-A-T-G-G-C-A-C-R-T-G-T-A-T-A-C-A-T-A-Y-G-T-A-A-C;

G-G-G-G-A-C-T-A-G-T-A-A-A-A-C-G-A-C-G-G-C-C-A-G-T;

C-C-G-G-C-A-T-C-G-A-T-R-R-R-R-R-R-C-G-A-C-G-G-C-C-A-G;

and G-A-G-A-G-G-A-G-A-A-G-G-A-G-A-G-A-G-A-A-R-R-R-R-R;

wherein R is either A or G and Y is either C or T.

5. The method of claim 1 wherein the primer has a sequence of T-G-T-G-T-G at its 3'"'"'-terminus.

6. The method of claim 1 wherein the primer is about 20 bases in length with a sequence of A-C-G-C-G-C-A-C at its 3'"'"'-terminus.

7. The method of claim 1 wherein the primer is at least about 17 bases in length and less than about 40 bases in length.

8. The method of claim 1 wherein said low stringency hybridization temperature is in the range of 35°

C. to 55°

C.

10. The method of claim 1 wherein said high stringency hybridization temperature in greater than about 55°

C.

11. The method of claim 1 wherein said high stringency hybridization temperature is about 60°

C.

12. The method of claim 1 wherein said low stringency hybridization temperature is about 40°

C. to about 50°

C., and said high stringency hybridization temperature is about 60°

C.

13. The method of claim 1 wherein said PCR thermocycle of step (b) is 2 PCR thermocycles.

14. The method of claim 13, wherein said plurality of PCR thermocycles is about 10 to 40 PCR thermocycles.

15. The method of claim 13 wherein said plurality of PCR thermocycles of step (d) is 10 PCR thermocycles.

9. The method of claim herein said low stringency hybridization temperature is about 40°

C. to about 48°

C.

16. A method for determining the species of a Staphylococcus bacteria, which method comprises:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA from a sample of Staphylococcus bacteria and at least one 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 a low stringency hybridization temperature permitting the arbitrary priming of said genomic DNA, thereby producing a set of discrete DNA segments;

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

(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 a high stringency hybridization temperature which does not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments;

(e) size-separating said amplified set of discrete DNA segments into bands to form a fingerprint of segments characteristic of said genome; and

(f) comparing said fingerprint of step (e) with the fingerprint for a control sample of genomic DNA from a predetermined species of Staphylococcus prepared in accordance with steps (a)-(e), thereby determining if said sample is the same species as said predetermined species.

17. The method of claim 16 wherein the control sample of Staphylococcus species is selected from the group consisting of S. haemolyticus, S. hominis, S. aureus, S. warneri, and S. cohnii.

18. The method of claim 16 wherein the primer is selected from the group consisting of

T-T-G-T-A-A-A-A-C-G-A-G-G-C-C-A-G-T;

G-G-A-A-A-C-A-G-C-T-A-T-G-A-C-C-A-T-G-A;

G-T-A-A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G;

G-C-A-A-T-T-A-A-C-C-C-T-C-A-C-T-A-A-A-G;

C-C-A-G-C-T-C-G-A-C-A-T-G-G-C-A-C-R-T-G-T-A-T-A-C-A-T-A-Y-G-T-A-A-C;

G-G-G-G-A-C-T-A-G-T-A-A-A-A-C-G-A-C-G-G-C-C-A-G-T;

G-A-G-A-G-G-A-G-A-A-G-G-A-G-A-G-A-G-A-A-R-R-R-R-R; and

C-C-G-G-C-A-T-C-G-A-T-R-R-R-R-R-R-C-G-A-C-G-G-G-C-A-G;

wherein R is either A or G and Y is either C or T.

19. The method of claim 16 wherein the primer has the sequence

C-C-A-G-C-T-C-G-A-C-A-T-G-G-C-A-C-R-T-G-T-A-T-A-C-A-T-A-Y- G-T-A-A-C, wherein R is either A or G and Y is either C or T.

20. A method for determining the species of a Streptococcus bacteria, which method comprises:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA from a sample of Streptococcus bacteria and at least one 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 a low stringency hybridization temperature permitting the arbitrary priming of said genomic DNA, thereby producing a set of discrete DNA segments;

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

(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 a high stringency hybridization temperature which does not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments;

(e) size-separating said amplified set of discrete DNA segments into bands to form a fingerprint of segments characteristic of said genome; and

(f) comparing said fingerprint of step (e) with the fingerprint for a control sample of genomic DNA from a predetermined species of Streptococcus prepared in accordance with steps (a)-(e), thereby determining if said sample is the same species as said predetermined species.

21. The method of claim 20 wherein the primer is selected from the group consisting of

T-T-G-T-A-A-A-A-C-G-A-G-G-C-C-A-G-T;

G-G-A-A-A-C-A-G-C-T-A-T-G-A-C-C-A-T-G-A;

G-T-A-A-T-A-C-G-A-C-T-C-A-C-T-A-T-A-G;

G-C-A-A-T-T-A-A-C-C-C-T-C-A-C-T-A-A-A-G;

C-C-A-G-C-T-C-G-A-C-A-T-G-G-C-A-C-R-T-G-T-A-T-A-C-A-T-A-Y-G-T-A-A-C;

G-G-G-G-A-C-T-A-G-T-A-A-A-A-C-G-A-C-G-G-C-C-A-G-T;

G-A-G-A-G-G-A-G-A-A-G-G-A-G-A-G-A-G-A-A-R-R-R-R-R; and

C-C-G-G-C-A-T-C-G-A-T-R-R-R-R-R-R-C-G-A-C-G-G-G-C-A-G;

wherein R is either A or G and Y is either C or T.

22. The method of claim 20 wherein the primer has the sequence C-C-A-G-C-T-C-G-A-C-A-T-G-G-C-A-C-R-T-G-T-A-T-A-C-A-T-A-Y- G-T-A-A-C, wherein R is either A or G and Y is either C or T.

23. A method for typing a bacterial isolate, which method comprises:

(a) forming a polymerase chain reaction (PCR) admixture by combining, in a PCR buffer, genomic DNA from a sample of bacteria and at least one 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 a low stringency hybridization temperature permitting the arbitrary priming of said genomic DNA, thereby producing a set of discrete DNA segments;

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

(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 a high stringency hybridization temperature which does not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments;

(e) size-separating said amplified set of discrete DNA segments into bands to form a fingerprint of segments characteristic of said genome; and

(f) comparing said fingerprint of step (e) with the fingerprints for control samples of genomic DNA from a panel of predetermined species of bacteria prepared in accordance with the steps (a)-(e), thereby determining if said sample is the same species as said predetermined species.

24. The method of claim 23 wherein the bacterial isolate belongs to the genus Staphylococcus and the panel of predetermined bacterial species comprises S. haemolyticus, S. hominis, S. aureus, S. warneri and S. cohnii.

25. A method of 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 polynucleotide 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 hybrid DNA-RNA molecule and at least one polynucleotide primer from about 10 to about 50 nucleotide bases in length;

(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 a low stringency hybridization temperature permitting the arbitrary priming of said hybrid DNA-RNA molecule, thereby producing said set of discrete DNA segments;

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

(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 a high stringency hybridization temperature which does not permit the formation of primer-template duplexes with a substantial degree of mismatching, thereby amplifying said set of discrete DNA segments.

26. The method of claim 25 further comprising the step of:

(g) size-separating said amplified set of discrete DNA segments into bands to form a fingerprint of segments characteristic of said single stranded RNA.