Length determination of nucleic acid repeat sequences by discontinuous primer extension

• US 20070178519A1
• Filed: 03/30/2007
• Published: 08/02/2007
• Est. Priority Date: 05/27/1997
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Livak, Kenneth, Lowe, Adam, Blasband, Andrew
• Granted Patent: US 7,414,115 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2535/125 Allele specific primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: C12Q 2565/515 characterised by the interaction between or sequential use of two or more arrays

Abstract

Disclosed is a method for determining the number of repeat units in a repeat region of a target nucleic acid. In a first aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer extension reagent; separating the target-primer hybrid and unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent; measuring a signal produced by the label; treating the label so as to render the label undetectable; and repeating the above steps until the signal is substantially less than a signal detected in a previous cycle. In a second aspect, the method of the invention includes the steps of annealing a primer to a target nucleic acid; performing a first primer extension reaction using a first primer-extension reagent; separating the target-primer hybrid from unreacted first primer extension reagent; performing a second primer extension reaction using a second primer extension reagent and with a primer termination reagent, the primer termination reagent including a nucleotide terminator having a label attached thereto; separating the target-primer hybrid from unreacted second primer extension reagent and unreacted primer termination reagent; measuring a signal produced by the label; and repeating the above steps until a signal is detected indicating incorporation of the nucleotide terminator. The invention further includes kits useful for practicing the above methods.

Claims

1-28. -28. (canceled)

29. A kit useful for determining the number of repeat units in a repeat region of a target nucleic acid comprising:

a plurality of different-sequence primers, each containing (i) a target binding segment and (ii) a tag segment having a nucleotide sequence that uniquely identifies the target binding segment, a first primer extension reagent; and

a second primer extension reagent, wherein at least one of the first or second primer extension reagents includes an extendible nucleotide having a photodestructible label attached thereto.

30. The kit of claim 29, which further includes an addressable array of immobilized, different tag complements, wherein each different tag complement contains a sequence that is complementary to one of said primer tag segments, under conditions effective to hybridize the tag segments to corresponding tag complements on the support.

31. The kit of claim 29 wherein the label comprises a fluorescent molecule.

32. The kit of claim 29 wherein the label is attached to the extendible nucleotide through a cleavable linker.

33. The kit of claim 29, wherein the second primer extension reagent includes a nucleotide terminator having a label attached thereto.

34. The kit of claim 33, wherein the nucleotide terminator comprises a label selected from the group consisting of a fluorescent molecule and a chemiluminescent molecule.

35. The kit of claim 33, wherein the nucleotide terminator comprises a photodestructible label.

36. The kit of claim 29, further comprising a polymerase.

37. The kit of claim 36, wherein the polymerase is Taq DNA polymerase.