Allele specific primer extension

• US 20040048301A1
• Filed: 08/29/2003
• Published: 03/11/2004
• Est. Priority Date: 08/29/2001
• Status: Active Grant

• Current Assignee: Global Life Sciences Solutions USA LLC (Danaher Corporation)
• Original Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Inventors: MacKlin, John, Nelson, John, Sood, Anup, Kumar, Shiv, Fuller, Carl
• Granted Patent: US 7,560,254 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 characterizing a nucleic acid template comprising the steps of:

(a) reacting said nucleic acid template, an allele specific primer, at least one terminal phosphate-labeled nucleotide and a DNA polymerase to produce a labeled polyphosphate;

(b) detecting said labeled polyphosphate; and

(c) characterizing said nucleic acid template.

2. The method of claim 1, wherein said detecting step includes (a) mixing said labeled polyphosphate with a phosphatase to produce a detectable species and (b) detecting said detectable species.

3. The method of claim 2, wherein said reacting step and said mixing step are carried out simultaneously.

4. The method of claim 2, wherein said detectable species is detectable by a property selected from the group consisting of color, fluorescence emission, chemiluminescence, mass change, oxidation/reduction potential and combinations thereof.

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

→

5′

exonuclease activity.

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

-most phosphodiester linkage.

7. The method of claim 1, further comprising the step of calculating the amount of said nucleic acid template from the quantity of labeled polyphosphate produced.

8. The method of claim 1, wherein one or more additional detection reagents are added in said reaction in said reacting step, and said additional detection reagents are detectably different from said labeled polyphosphate.

9. The method of claim 1, wherein said reaction in said reacting step includes two or more terminal phosphate-labeled nucleotides.

10. The method of claim 9, wherein said two or more terminal phosphate-labeled nucleotides include enzyme-activatable labels selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

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

-end base.

12. The method of claim 1, wherein said terminal phosphate-labeled nucleotide comprises four or more phosphate groups in the polyphosphate chain.

13. 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.

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

→

5′

exonuclease activity and said allele specific primer is nuclease resistant.

15. The method of claim 1, wherein said nucleic acid template is polymorphic at the position complementary to the 3′

-base of said allele specific primer, and the presence of labeled polyphosphate reveals the identity of the nucleotide base of said nucleic acid template at said position.

16. The method of claim 1, wherein said terminal-phosphate-labeled nucleotide is:

17. The method of claim 16, 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.

18. The method of claim 16, 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.

19. The method of claim 16, wherein said enzyme-activatable label is selected from the group consisting of chemiluminescent compounds, fluorogenic dyes, chromogenic dyes, mass tags, electrochemical tags and combinations thereof.

20. The method of claim 19, wherein said enzyme-activatable label is a chromogenic moiety selected from the group consisting of 5-bromo-4-chloro-3-indolyl phosphate, 3-indoxyl phosphate, p-nitrophenyl phosphate, and derivatives thereof.

21. The method of claim 19, wherein said enzyme-activable label is a phosphorylated label and a fluorogenic moiety 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.

22. The method of claim 19, wherein said label is an alkaline phosphatase-activated 1,2-dioxetane compound.

23. The method of claim 22, wherein said 1,2-dioxetane compound is 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.

28. The method of claim 1, wherein multiple cycles of nucleic acid polymerase reaction are performed during said reacting step, whereas reaction products are thermally separated from said nucleic acid template at the end of each cycle.

24. A kit for detection of a polymorphism of a specific nucleotide base in a target nucleic acid chain comprising:

(a) at least one terminal-phosphate-labeled nucleotide;

(b) a DNA polymerase; and

a

(c) a phosphatase.

25. The kit of claim 24, further comprising a nuclease with enzymatic activity sufficient to decompose DNA in the 3′

→

5′

direction.

26. The kit of claim 24, further comprising a nuclease resistant oligonucleotide primer having a sequence complimentary to at least a part of said target polynucleotide.

27. The kit of claim 24, wherein said DNA polymerase has enzymatic activity sufficient to decompose DNA in a 3′

→

5′

direction.