Reagent for nucleic acid typing by primer extension

• US 6,537,748 B1
• Filed: 02/26/1999
• Issued: 03/25/2003
• Est. Priority Date: 03/05/1991
• Status: Expired due to Term

• Current Assignee: Laboratory Corporation Of America Holdings
• Original Assignee: Orchid Biosciences Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Anderson, Stephen, Knapp, Michael R., Goelet, Philip
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/258,133
• Publication Number:
  US 20030044779A1
• Time in Patent Office: 1,488 Days
• Field of Search: 435/6, 435/91.2, 435/5, 536/23.1, 536/26.1, 536/24.33, 536/26.12
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 2525/121 incorporating both deoxyribonucleotides and ribonucleotides
• CPC Class Codes: C12Q 2535/101 Sanger sequencing method, i.e. oligonucleotide sequencing using primer elongation and dideoxynucleotides as chain terminators
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

This invention concerns a reagent composition comprising at least two different terminators of a nucleic acid template-dependent, primer extension reaction. This invention also concerns a method for determining the identity of a nucleotide base at a specific position in a nucleic acid of interest. This invention further concerns a method for determining the presence or absence of a particular nucleotide sequence in a sample of nucleic acids. This invention further concerns a method for identifying different alleles in a sample containing nucleic acids. This invention further concerns a method for determining the genotype of an organism at one or more particular genetic loci.

Claims

1. A reagent composition which comprises an aqueous carrier, a oligonucleotide primer molecule, a DNA polymerase, and an admixture of at least two different nucleotide terminators, or terminator nucleotide analogs, of a nucleic acid template dependent, primer extension reaction, each of the terminators being capable of specifically terminating the extension reaction and at least one of the terminators being labeled with a detectable marker, and wherein the reagent composition lacks dATP, dCTP, dGTP and dTTP.

2. A reagent of claim 1, wherein the reagent comprises four different terminators.

3. A reagent of claim 2, wherein two of the terminators are labeled, each with a different detectable marker.

4. A reagent of claim 2, wherein three of the terminators are labeled, each with a different detectable marker.

5. A reagent of claim 2, wherein the four terminators are labeled, each with a different detectable marker.

6. A reagent of any of claim 1-5, wherein the terminators comprise dideoxynucleotides.

7. A reagent of claim 6, wherein the terminators comprise one or more of ddATP, ddCTP, ddGTP, ddTTP or ddUTP.

8. A reagent of any of claims 1-5, wherein the terminators comprise arabinoside triphosphates.

9. A reagent of any of claims 1-5, wherein each of the detectable markers is an isotopically labeled moiety, a chromophore, a fluorophore, a protein moiety, or a moiety to which an isotopically labeled moiety, a chromophore, a fluorophore, or a protein moiety can be attached.

10. A reagent of any of claims 3-5, wherein each of the different detectable markers is a different fluorophore.

11. A reagent of any of claims 1-5, wherein the reagent further comprises pyrophosphatase.