Method for profiling nucleic acids of unknown sequence using arbitrary oligonucleotide primers

• US 5,413,909 A
• Filed: 01/19/1993
• Issued: 05/09/1995
• Est. Priority Date: 08/24/1990
• Status: Expired due to Fees

• Current Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Original Assignee: The University Of Tennessee Research Corporation (University of Tennessee)
• Inventors: Gresshoff, Peter M., Caetano-Anolles, Gustavo, Bassam, Brant J.
• Primary Examiner(s): Parr, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/006,380
• Time in Patent Office: 840 Days
• Field of Search: 435/6, 435/91, 435/91.1, 435/91.2, 436/501, 536/22.1-24.33, 935/77, 935/88, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2535/139 Random amplification polymorphism detection [RAPD]

Abstract

A method for amplifying nucleic acid sequences in a template comprising at least one nucleic acid or mixture of nucleic acids wherein each nucleic acid comprises at least one strand. The method comprises the steps of treating the template with at least one oligonucleotide primer having a nucleic acid sequence that is substantially complementary to at least one nucleic acid sequence on the template under conditions such that the primer will bind to a segment that is substantially complementary to the primer, producing primed strands of nucleic acid. The primed strands produce extension strands, wherein the extension strands comprise the primer in combination with a sequence of nucleic acids that is substantially complementary to the template in a 5'"'"' to 3'"'"' direction from the primer. The extension strands are further treated with the primer producing amplification strands which are treated with primer. The amplification strands are repeatedly treated with primer to produce large numbers of amplification strands whereby the nucleic acid sequences of the amplification strands are substantially similar to nucleic acid sequences in the original template.

Claims

1. A method for generating a pattern characteristic of at least one nucleic acid of unspecified sequence in a sample, which method comprises:

a) treating said nucleic acid with at least one oligonucleotide primer having an arbitrary sequence and of 5 to 25 nucleotides in length, the amount of primer being in excess over the amount of said nucleic acid, the amounts being in mass and allowing said primer to anneal to multiple sites on each strand of said nucleic acid, each site being substantially complementary to the nucleotides of which said primer is constituted, thus forming a multiplicity of a set of primed templates,

b) treating said primed templates with a nucleic acid polymerase, thereby generating a multiplicity of extension strands, the extension strands comprising the primer in combination with a sequence of nucleotides that is substantially complementary to the templates, and extending along the template strands to either the 5'"'"' end of the template strands or the next primed site on the template, the number of extension strands thereby corresponding substantially to the number of primed sites,

c) denaturing the extension strands from the template strands, thereby generating corresponding single-stranded extension strands and templates,

d) allowing said primer to anneal to the extension strands and to the template strands,

e) repeating steps (a), (b), (c) and (d) until there are generated nucleic acid fragments which are characteristic and unique for said nucleic acid of unspecified sequence,

f) separating said fragments, and

g) determining the characteristic pattern of fragments generated from said sequence of nucleic acid in the sample.

2. The method of claim 1 which comprises comparing the pattern resulting from step (g) with the pattern of fragments from another sequence of nucleic acid generated in (g) using a primer of the same sequence, thereby determining any difference and/or similarity between the two patterns of fragments by size.

3. The method of claim 1 wherein the primer is selected from the group consisting of primers of 5 to 10 nucleotides in length.

4. The method of claim 3 wherein the primer is selected from a group consisting of primers of 5, 8, 9 and 10 nucleotides in length.

5. The method of claim 2 wherein the sequence of the primer is known.

6. The method of claim 2 wherein the amount of primer is from 50,000 to 100,000 times that of the nucleic acids.

7. The method of claim 2 wherein the sample is a sample of mammalian tissue.

8. The method of claim 2 wherein the sample is a sample of plant tissue.

9. The method of claim 2 wherein the identification of the pattern is performed on a polyacrylamide gel.

17. The method of claim 1 which comprises treating the generated nucleic acid fragments with a restriction enzyme to produce subfragments of said nucleic acid fragments.

10. A method for generating a pattern characteristic of at least one nucleic acid of unspecified sequence in a sample which method comprises:

a) treating said nucleic acid with at least one oligonucleotide primer having an arbitrary sequence and of 8 nucleotides in length, the amount of primer being in excess over the amount of said nucleic acid, the amounts being in mass, and allowing said primer to anneal to multiple sites on each strand of said nucleic acid, each site being substantially complementary to the nucleotides of which said primer is constituted, thus forming a multiplicity of a set of primed templates,

b) treating said primed templates with a nucleic acid polymerase, thereby generating a multiplicity of extension strands, the extension strands comprising the primer in combination with a sequence of nucleotides that is substantially complementary to the templates, and extending along the template strands to either the 5'"'"' end of the template strands or the next primed site on the template, the number of extension strands thereby corresponding substantially to the number of primed sites,

c) denaturing the extension strands from the template strands thereby generating corresponding single-stranded extension strands and templates,

d) allowing said primer to anneal to the extensions strands and to the template strands,

(e) repeating steps (a), (b), (c) and (d) until there are generated nucleic acid fragments which are characteristic and unique for said nucleic acid of unspecified sequence,

f) separating said fragments, and

g) determining the characteristic pattern of fragments generated from said sequence of nucleic acid in the sample.

11. The method of claim 10 wherein the amount of primer is from 50,000 to 100,000 times that of the nucleic acids.

12. The method of claim 10 wherein the sequence of the primer is known.

13. The method of claim 10 which comprises comparing the pattern resulting from step (g) with the pattern of fragments from another sequence of nucleic acid generated in (g) using a primer of the same sequence, thereby determining any difference and/or similarity between the two patterns of fragments by size.

14. The method of claim 13 wherein the determination of the characteristic pattern is performed on a polyacrylamide gel.

15. The method of claim 13 wherein the sample is a sample of mammalian tissue.

16. The method of claim 13 wherein the sample is a sample of plant tissue.

18. The method of claim 10 which comprises treating the generated nucleic acid fragments with a restriction enzyme to produce subfragments of said nucleic acid fragments.

19. The method of claim 16 wherein the comparison is of two patterns generated for the nucleic acid from plant tissue from two different cultivars of the same plant species.

20. The method of claim 13 wherein the sample is a bacteriophage.