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Methods and assays for capture of nucleic acids

A nucleic acid and nucleotide technology used in the field of nucleic acid capture and assay

Inactive Publication Date: 2011-05-04
F HOFFMANN LA ROCHE & CO AG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]However, the method used has low signal-to-noise ratio and reproducibility issues

Method used

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  • Methods and assays for capture of nucleic acids
  • Methods and assays for capture of nucleic acids
  • Methods and assays for capture of nucleic acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Target Capture of Creatine Phosphokinase 6 (CPK6) and Restriction Enzyme Digested PCR Amplicons

[0084] Experiments were designed to test ligation efficiency in the methods and assays of the invention. Experiments were designed using 3'-labeled CPK6 oligonucleotides (Integrated DNA Technologies (IDT), Coralville IA) and PCR fragments amplified from E. coli genomic DNA (ATCC No. 700926D-5), yielding from about 1100 to about 1500 bases Base pair amplicons (SEQ ID NO: 2, 3 and 4). 50- and 60-mer oligonucleotide probes containing 16 base pair hairpins were designed. The hairpin sequence used is 5'-CCGGAGGATACTCCG G-3' (SEQ ID NO: 1), such as image 3 shown. A control probe was synthesized which did not contain a hairpin structure. Quadrants of probes were designed representing all 4 bases of each strand, such that a total of 8 probes were designed for target nucleotides within the query sequence (e.g., 4 per strand of the DNA target ). With the exception of the termi...

Embodiment 2

[0092] Target capture of creatine phosphokinase 6 (CPK6) and PCR amplicons

[0093] Experiments were designed to test cap endonucleases and ligation efficiency in the methods and assays of the invention. Experiments were designed as described in Example 1 using 3'-labeled CPK6 oligonucleotides (IDT) and PCR fragments amplified from E. coli genomic DNA. 50- and 60-mer oligonucleotide probes were designed containing a 16 base pair hairpin and complementary bases at the terminal 3' end. The hairpin sequence used was . A control probe was synthesized which did not contain a hairpin structure. Four sets of probes were designed, such as Figure 1A As shown, which represent all 4 bases of each strand, such that a total of 8 probes were designed for target nucleotides within the query sequence (eg, 4 / each strand of the DNA target). Probes were synthesized in situ on the array at Roche NimbleGen (Madison, WI) at a density of 2.1 million probes / array (HD2).

[0094] Three PCR f...

Embodiment 3

[0098] Creatine Phosphokinase 6 (CPK6) and Target Capture of Randomly Fragmented Genomic DNA

[0099] Experiments were designed using human and E. coli genomic DNA (gDNA). Oligonucleotide probes were designed as described in Example 1 to synthesize probes in situ on a Roche NimbleGen (Madison, WI) at a density of 2.1 million probes / array (HD2). Fragmentation of genomic DNA (gDNA) by sonication or random amplification of genomic DNA (gDNA) using Klenow fragments using random primers of different lengths (9mer, 10mer, 12mer, and 15mer) , to obtain amplified target sequences of different lengths. Fragments were treated with Antarctic phosphatase, labeled at their 3' ends with Cy3-ddCTP using terminal transferase (TdT), and unlabeled fragments were precipitated using methods known to those skilled in the art. Microarray slides were sealed with a HX3 mixer (NimbleGen Roche), and denatured, labeled target sequences were applied to the microarray (5-30 μg sample / subarray). Probes ...

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Abstract

The present disclosure provides methods and systems for sequence specific nucleic acid target capture comprising enzymatic reactions. The present disclosure relates to a plurality of oligonucleotide probes for capture and subsequent detection of target nucleic acid sequences, using flap endonucleases, ligases, and / or additional enzymes, proteins or compounds, on substrates, for example microarray slides, and in solution formats.

Description

field of invention [0001] The present invention provides sequence-specific nucleic acid target capture methods and systems involving enzymatic reactions. More specifically, the invention encompasses the capture and subsequent detection of target nucleic acid sequences on a substrate (e.g., a microarray slide) and in solution using a cap endonuclease, ligase, and / or other enzyme, protein, or compound. multiple oligonucleotide probes. Background of the invention [0002] The advent of nucleic acid microarray technology has made it possible to create arrays of millions of nucleic acid sequences in a very small area, eg, on a microscope slide (eg, US 6,375,903 and US 5,143,854). Initially, such arrays were created by blotting pre-synthesized DNA sequences onto glass slides. However, the construction of a maskless array synthesizer (MAS) as described in US 6,375,903 now allows the in situ synthesis of oligonucleotide sequences directly on the glass slide itself. [0003] Usin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6837C12Q2525/301C12Q2521/501C12Q2521/301C12Q2561/109
Inventor T·艾伯特V·拉米彻夫J·帕特尔
Owner F HOFFMANN LA ROCHE & CO AG