Process for detecting polymorphisms on the basis of nucleotide differences

• US 5,126,239 A
• Filed: 03/14/1990
• Issued: 06/30/1992
• Est. Priority Date: 03/14/1990
• Status: Expired due to Term

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Tingey, Scott V., Livak, Kenneth J., Williams, John G., Rafalski, Jan A.
• Primary Examiner(s): Wax, Robert A.
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/494,258
• Time in Patent Office: 839 Days
• Field of Search: 435/6, 435/91, 436/501, 436/94, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

A process is provided for detecting polymorphisms on the basis of nucleotide differences in random segments of the nucleic acid by performing a primer extension reaction on the nucleic acids and comparing the extension reaction products. The random nucleic acid segment can be amplified by first dissociating the extension product from the template and contacting the dissociated extension product with a primer under conditions such that an amplification extension product is synthesized using the dissociated extension product as a template. Differences in the extension product are useful as markers in constructing genetic maps and as markers to distinguish or identify individuals.

Claims

1. A process for detecting polymorphisms on the basis of nucleotide differences in random segments of nucleic acids comprising:

(a) separately performing an extension reaction on a random segment of each of at least two nucleic acids from different sources, said reaction comprising;

(i) contacting each of the nucleic acids with at least one nonspecifically targeted oligonucleotide primer of greater than 7 nucleotides under conditions such that for at least one nucleic acid a random extension product of at least one primer is synthesized; and

(b) comparing the results of the separately performed random extension reactions for differences.

2. The process of claim 1, after step (i) and before step (b), further comprising the additional steps of (ii) dissociating the extension product from its complement;

and (iii) amplifying the random nucleic acid segment by contacting the dissociated extension product with at least one primer of step (i) under conditions such that an amplification extension product is synthesized using the dissociated extension product as a template.

3. The process of claim 2 wherein the steps are performed at least twice.

4. The process of claim 1 or 2 using a nucleic acid polymers and nucleoside triphosphate substrates or their analogues and mixtures thereof.

5. The process of claim 1 or 2 wherein one primer is employed.

6. The process of claim 1 or 2 wherein the primer is from 9 to 10 nucleotides in length.

7. The process of claim 1 or 2 wherein at least one primer is biotinylated.

8. The process of claim 1 or 2 wherein the nucleic acids are from different individual organisms.

9. A process according to claim 4, wherein the nucleic acid polymerase is a DNA polymerase and the nucleoside triphosphate substrates are deoxyribonucleoside triphosphate substrates.

10. A process according to claim 9, wherein the DNA polymerase is a thermostable polymerase.

11. A process according to claim 10, wherein the DNA polymerase is Taq polymerase.

12. The process of claim 1 or 2, wherein the comparison is through size separation of the random extension products.

13. A process according to claim 12, wherein the size separation comparison is performed by electrophoresis through a polyacrylamide gel matrix or agarose gel matrix.

14. The process of claim 1 or 2, wherein the comparison is through nucleotide sequence determination.

15. The process of claim 1 or 2, wherein the nucleic acid source is selected from the group consisting of plants, animals and microbes.

16. The process of claim 1 or 2 wherein the nucleic acid source is human.

17. The process of claim 1 or 2 wherein a difference in the random extension products is used as a marker to construct a genetic map.

18. The process of claim 1 or 2 wherein a difference in the random extension products is used as a marker to distinguish or identify individuals.