Methods of labeling nucleic acids for use in array based hybridization assays

a nucleic acid and array technology, applied in the field of nucleic acid labeling, can solve the problems of limited spectrum of fluorescent labels that may be employed in protocols where labeled nucleotide analogs are generated from labeled nucleotide analogs, and not per

Inactive Publication Date: 2001-07-12
TAKARA BIO USA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

While the above approaches are effective in many situations, they are not perfect.
For example, the spectrum of fluorescent labels that may be employed in protocols where labeled targets are generated from labeled nucleotide analogs is limited, as not all fluorescently tagged nucleotide analogs can be processed by enzymes, e.g. polymerases, that are employed in the labeled target generation step.

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  • Methods of labeling nucleic acids for use in array based hybridization assays
  • Methods of labeling nucleic acids for use in array based hybridization assays
  • Methods of labeling nucleic acids for use in array based hybridization assays

Examples

Experimental program
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example 1

[0051] Preparation of Cy3 labeled target nucleic acid.

[0052] 1. To 5 .mu.g of polyA.sup.+ placental RNA add 5 .mu.l of gene specific primers (0.2 .mu.M each as described in PCT / US98 / 10561 the disclosure of which is herein incorporated by reference). Add water to total final volume of 25 .mu.l.

[0053] 2. Heat at 70.degree. C. for 5 min.

[0054] 3. Cool to 48.degree. C., add 25 .mu.l of following master mix:

1 5 .times. first strand buffer 10 .mu.l 10 .times. dNTP mixture 5 .mu.l MMLV RT (200 u / ml) 2.5 .mu.l Milli Q water 7.5 .mu.l

[0055]

2 Composition of 10 .times. dNTP mixture for 100 .mu.l Reagent Stock conc. Amount Final conc. DATP 100 mM 5 .mu.l 5 mM DCTP 100 mM 5 .mu.l 5 mM DGTP 100 mM 5 .mu.l 5 mM DTTP 100 mM 2.5 .mu.l 2.5 mM Allylamino-dUTP 10 mM 25 .mu.l 2.5 mM Milli Q water 57.5 .mu.l

[0056] 4. Incubate at 48.degree. C. for 30 min.

[0057] 5. Heat to 70.degree. C. for 5 min.

[0058] 6. Cool to 37.degree. C. and add 0.5 .mu.l of RNase H (10 U / .mu.l)

[0059] 7. Incubate at 37.degree. C. fo...

example 2

[0079] Post Hybridization Labeling

[0080] Target nucleic acids are prepared as described above, except that the amino functional group is replaced with an SH group. The target nucleic acids are then hybridized to an array of probe nucleic acids. Following hybridization, the array surface is washed to removed unhybridized target. The array surface is then contacted with a solution of maleimide functionalized label under conditions sufficient for the functionalized label to conjugate to the hybridized target via the SH moiety, resulting in an array of labeled hybridized targets.

[0081] It is evident that the subject invention provides an important new method for generating labeled target nucleic acids for use in array based hybridization assays. With the subject methods, one is not limited to using labeled analogs that can be processed by polymerases, since the labeling step occurs after the target generation step. As such, the number of different types of labels that can now be used in...

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Abstract

Methods and kits are provided for labeling nucleic acids, e.g. for use in array based hybridization assays. In the subject methods, target nucleic acid is generated from an initial nucleic acid source, e.g. mRNA, where the target nucleic acid is characterized by having at least one reactive functionality that is not a moiety found on naturally occurring nucleic acids. Functionalized label is then conjugated to the target nucleic acid, either before or after it has been hybridized to array of nucleic acids stably associated with the surface of a solid support. The subject methods find use in a variety of array based hybridization assays, including differential expression assays.

Description

[0001] This application is a continuation-in-part of application Ser. No. ______ filed Nov. 19, 1999, which application claims priority to application Ser. No. PCT / US98 / 10561 filed on May 21, 1998; which claims priority to application Ser. No. 09 / 053,375 filed on Mar. 31, 1998 and application Ser. No. 08 / 859,998 filed on May 21, 1997, the disclosures of which are herein incorporated by reference.[0002] 1. Field of the Invention[0003] The field of the invention is nucleic acid labeling, particularly labeling of nucleic acid targets for use in array based hybridization assays.[0004] 2. Background of the Invention[0005] Nucleic acid arrays have become an increasingly important tool in the biotechnology industry and related fields. Nucleic acid arrays, in which a plurality of nucleic acids are deposited onto a solid support surface in the form of an array or pattern, find use in a variety of applications, including drug screening, nucleic acid sequencing, mutation analysis, and the like...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68
CPCC12Q1/6816C12Q1/6837C12Q2565/101C12Q2525/197C12Q2525/101
Inventor BOCHKARIOV, DMITRY E.CHENCHIK, ALEX
Owner TAKARA BIO USA INC
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