Differentiation of nucleic acid segments on the basis of nucleotide differences

• US 4,879,214 A
• Filed: 11/15/1988
• Issued: 11/07/1989
• Est. Priority Date: 11/15/1988
• Status: Expired due to Fees

• Current Assignee: PerkinElmer Life Sciences, Inc.
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Kornher, John S., Livak, Kenneth J.
• Primary Examiner(s): Lilling, Herbert J.
• Assistant Examiner(s): Zitomer, Stephanie W.
• Application Number:
  US07/272,068
• Time in Patent Office: 357 Days
• Field of Search: 435/6, 435/91, 935/1, 935/2, 935/3, 935/8, 935/17, 935/18, 935/76, 935/77
• US Class Current: 435/6.18
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

A process for distinguishing nucleic acid segments on the basis of nucleotide differences, thereby providing a rapid, convenient means for detecting mutation, is disclosed. The present process comprises synthesizing separately complementary nucleic acid strands on each of at least two target nucleic acid templates using a nucleic acid polymerase and nucleoside triphosphate substrates, wherein at least one of the natural nucleoside triphosphate substrates is replaced with a mobility-shifting analog; denaturing the synthesized strands from the templates if necessary; and comparing the mobility of the separately synthesized strands through a size-fractionation medium. A difference in mobility indicates that the target nucleic acid templates contain different numbers of total nucleotides and/or different numbers of nucleotides complementary to the mobility-shifting analogs. Thus, the process of the present invention can be used to distinguish homologous DNAs or RNAs differing by nucleotide substitutions that affect the number of mobility-shifting analog residues incorporated into synthesized strands, as well as to distinguish differences due to nucleotide insertion or deletion.

Claims

1. A process for distinguishing nucleic acid segments on the basis of nucleotide differences, which comprises

synthesizing separately complementary nucleic acid strands on each of at least two target nucleic acid templates using a nucleic acid polymerase and nucleoside triphosphate substrates, wherein at least one of the natural nucleoside triphosphate substrates is completely replaced with a mobility-shifting analog;

and comparing the mobility of the separately synthesized strands in single-stranded form through a size-fractionation medium.

2. A process according to claim 1, wherein the nucleic acid polymerase is DNA polymerase and the nucleoside triphosphate substrates are deoxyribonucleoside triphosphate substrates.

3. A process according to claim 2 wherein the DNA polymerase is selected from the group consisting of Taq polymerase, E. coli DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, reverse transcriptase, phage T4 DNA polymerase, and phage T7 DNA polymerase.

4. A process according to claim 1 wherein the nucleic acid polymerase is RNA polymerase and the nucleoside triphosphate substrates are ribonucleoside triphosphate substrates.

5. A process according to claims 1, 2, or 4 wherein the synthesis of complementary nucleic acid strands is performed by a primer extension reaction.

6. A process according to claim 5 wherein the primer extension reaction is a polymerase chain reaction (PCR).

7. A process according to claims 1, 2, or 4 wherein the mobility-shifting analog is 5-(Bio-AC-AP3)dCTP.

8. A process according to claims 1, 2, or 4 wherein the comparison of mobility of the separately synthesized strands is performed by elecrophoresis through a polyacrylamide gel matrix.

9. A process according to claim 8 wherein the polyacrylamide gel matrix contains a denaturant.

10. A process according to claim 1 wherein the synthesized strands are denatured from the templates.