Allele specific primer extension

• US 7,560,254 B2
• Filed: 08/29/2003
• Issued: 07/14/2009
• Est. Priority Date: 08/29/2001
• Status: Expired due to Term

• Current Assignee: Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Inventors: Nelson, John, Macklin, John, Fuller, Carl, Sood, Anup, Kumar, Shiv
• Primary Examiner(s): Lu; Frank W
• Application Number:
  US10/651,558
• Publication Number:
  US 20040048301A1
• Time in Patent Office: 2,146 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 435/183, 436/94, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation

Abstract

A method of characterizing a nucleic acid sample is provided that includes the steps of: (a) conducting a DNA polymerase reaction that includes the reaction of a template, an allele specific primer, at least one terminal phosphate-labeled nucleotide, DNA polymerase, and optionally an enzyme having 3′→5′ exonuclease activity when the primer is non-hydrolyzable, which reaction results in the production of labeled polyphosphate; (b) permitting the labeled polyphosphate to react with a phosphatase to produce a detectable species; (c) detecting the detectable species; and (d) characterizing the nucleic acid sample based on such detection.

Claims

1. A method of detecting a polymorphic site of a nucleic acid template comprising the steps of:

(a) conducting a nucleic acid polymerase reaction in a solution comprising a nucleic acid template which contains a polymorphic site, an allele specific primer whose 3′

terminal base is complementary to the nucleotide of the polymorphic site of said nucleic acid template, two or more terminal phosphate-labeled nucleotides which are non-reactive to phosphatases, and a DNA polymerase so that a labeled polyphosphate is produced;

(b) mixing a phosphatase with said solution in step (a) and producing a labeled detectable species from said labeled polyphosphate;

(c) detecting said labeled detectable species wherein the detection of said labeled detectable species indicates the presence of the polymorphic site in said nucleic acid template.

2. The method of claim 1, wherein the steps (a) and (b) are carried out simultaneously.

3. The method of claim 1, wherein the label in said terminal phosphate-labeled nucleotides is selected from the group consisting of an electrochemical label, a mass tag, a chromogenic dye label, chemiluminescent dye label, and fluorescent dye label.

4. The method of claim 1, wherein said allele specific primer is nuclease resistant and said conducting step further includes an enzyme having 3′

→

5′

exonuclease activity.

5. The method of claim 4, wherein said nuclease resistant primer has a methyl phosphonate, a borano phosphate or a phosphorothioate linkage at the 3′

end of the primer.

6. The method of claim 1 further comprising adding one or more additional detection reagents in said conducting step, and said additional detection reagents are different from said labeled polyphosphate.

7. The method of claim 1, wherein said allele specific primer has a mismatch located at one, two or three bases away from the 3′

-end base of said allele specific primer.

8. The method of claim 1, wherein the polyphosphate chain of at least one of said terminal phosphate-labeled nucleotides comprises four, five or six phosphate groups.

9. The method of claim 1 wherein, said DNA polymerase is selected from the group consisting of the Klenow fragment of DNA polymerase I, Phi 29 DNA polymerase, DNA polymerase I, T4 DNA polymerase, Thermo Sequenase, Sequenase, Taq DNA polymerase, pfu DNA polymerase, Amplitaq FS, reverse transcriptase and T7 DNA polymerase.

10. The method of claim 1, wherein said DNA polymerase has 3′

→

5′

exonuclease activity and said allele specific primer is nuclease resistant.

11. The method of claim 1, wherein each of said two or more terminal phosphate-labeled nucleotides is:

12. The method of claim 11, wherein said sugar moiety is selected from the group consisting of ribosyl, 2′

-deoxyribosyl, 3′

-deoxyribosyl, 2′

,3′

-dideoxyribosyl, 2′

,3′

-didehydrodideoxyribosyl, 2′

-alkoxyribosyl, 2′

-azidoribosyl, 2′

-aminoribosyl, 2′

-fluororibosyl, 2′

-mercaptoriboxyl, 2′

-alkylthioribosyl, 3′

-alkoxyribosyl, 3′

-azidoribosyl, 3′

-aminoribosyl, 3′

-fluororibosyl, 3′

-mercaptoriboxyl, 3′

-alkylthioribosyl, carbocyclic, acyclic and other modified sugars.

13. The method of claim 11, wherein said base is selected from the group consisting of uracil, thymine, cytosine, guanine, 7-deazaguanine, hypoxanthine, 7-deazahypoxanthine, adenine, 7-deazaadenine, 2,6-diaminopurine and analogs thereof.

14. The method of claim 11, wherein said enzyme-activatable label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, and electrochemical tags.

15. The method of claim 14, wherein said chromogenic dyes are selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate, and derivatives thereof.

16. The method of claim 11, wherein said P-L in said terminal phosphate-labeled nucleotides is a phosphorylated label comprising a fluorogenic dye selected from the group consisting of 2-(5′

-chloro-2′

-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone, fluorescein diphosphate, fluorescein 3′

(6′

)-O-alkyl-6′

(3′

)-phosphate, 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl)phosphate, 4-methylumbelliferyl phosphate, resorufin phosphate, 4-trifluoromethylumbelliferyl phosphate, umbelliferyl phosphate, 3-cyanoumbelliferyl phosphate, 9,9-dimethylacirdin-2-one-7-yl phosphate, 6,8-difluoro-4-methylumbelliferyl phosphate, and derivatives thereof.

17. The method of claim 14, wherein said chemiluminescent compounds are alkaline phosphatase-activated 1,2-dioxetane compounds.

18. The method of claim 17, wherein said 1,2-dioxetane compounds are selected from the group consisting of 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,2′

-(5-chloro-)tricyclo[3,3,1-13,7]-decan]-1-yl)-1-phenyl phosphate, chloroadamant-2′

-ylidenemethoxyphenoxy phosphorylated dioxetane, 3-(2′

-spiroadamantane)-4-methoxy-4-(3″

-phosphoryloxy)phenyl-1,2-dioxetane and derivatives thereof.

19. The method of claim 1, wherein said nucleic acid polymerase reaction are performed for multiple cycles.