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Methods of nonspecific target capture of nucleic acids

a nucleic acid and target capture technology, applied in the field of molecular biology, can solve the problems of long time required to complete the nucleic acid isolation, complicated procedures, and add complexity to the design, optimization and performance of methods

Inactive Publication Date: 2013-08-15
GEN PROBE INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many methods involve complicated procedures, use harsh chemicals or conditions, or require a long time to complete the nucleic acid isolation.
Some methods involve use of specialized oligonucleotides, each specific for an intended target nucleic acid which adds complexity to the design, optimization and performance of methods, particularly if isolation of more than one target nucleic acid is desired or if the sequence of the desired target nucleic acid is unknown.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

Nonspecific Target Capture of RNA

[0045]This example demonstrates the efficiency of various nonspecific target capture probe to capture RNA. Tests were performed by using a known amount of Chlamydia trachomatis rRNA (a mixture of 16S and 23S rRNA) which was hybridized to a labeled detection probe complementary to a sequence in the rRNA to label the target rRNA before it was captured. Typically, to prepare the test samples, 200 fmole of rRNA was hybridized with 1 pmole of a AE-labeled oligonucleotide in hybridization reagent (60 C for 60 min in 0.04 ml), and then cooled to room temperature (RT) and diluted with 0.3 ml of hybridization reagent. An aliquot (10 μl) of the probe-labeled target nucleic acid mixture was mixed with 0.5 ml of a substantially aqueous solution (made up of 0.2 ml sample transport reagent, 0.2 ml water and, 0.1 ml of TCR containing 50 μg of magnetic particles with poly-dT immobilized probes) and 20 pmoles of the nonspecific capture probe. The mixture was incubate...

example 2

Nonspecific Target Capture of RNA

[0055]This example demonstrates efficiencies of nonspecific target capture of rRNA by using a variety of different capture probes in assays performed substantially as described in Example 1 except that the target capture incubations were at RT for 10 to 30 min. All of the nonspecific capture probes used were synthesized using RNA bases and 2′-methoxy linkages and a 3′ DNA tail sequence.

[0056]In a first set of tests, the C9-containing nonspecific capture probes of SEQ ID Nos. 5 and 6 were tested using incubation at RT for 30 min, and the efficiency of RNA capture was 71% for capture probe of SEQ ID NO:5 and 89.5% for the capture probe of SEQ ID NO:6, compared to the positive control of 53% capture and the negative control of 1.3% capture. These results shown that incubation at 60 C was not needed for efficient nonspecific target capture of RNA.

[0057]In a second set of target captures, also incubated at RT for 30 min, the nonspecific capture probe of S...

example 3

Target Capture of HIV-1 Target RNA

[0062]This example demonstrates the use of two different nonspecific target capture probes to capture HIV-1 sequences, which were synthetic RNA sequences corresponding to portions of the protease-encoding gene of and the RT4 gene of HIV-1. Tests were performed individually for both target nucleic acids by using a known amount of in vitro RNA transcripts prepared from cloned Protease and RT4 sequences (a 681 nt Protease transcript, and a 471 nt RT4 transcript) which were hybridized specifically and individually to a labeled detection probe complementary to a sequence in the target rRNA before it was captured. To prepare the test samples, 200 fmole of the target RNA was hybridized specifically with 1 pmole of a AE-labeled detection probe oligonucleotide in hybridization reagent (60 C for 60 min in 0.04 ml), cooled to room temperature (RT) and diluted with 0.3 ml of hybridization reagent. An aliquot (10 μl) of the probe-labeled target RNA mixture was m...

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Abstract

Methods for capturing a target nucleic acid from a sample by using a capture probe that binds nonspecifically to the target nucleic acid and binds specifically to an immobilized probe via a specific binding pair that has one member on the capture probe and one member on the immobilized probe are disclosed. Compositions that include a capture probe that binds nonspecifically to a target nucleic acid and specifically to an immobilized probe via binding of members of a specific binding pair in a solution phase of a reaction mixture are disclosed.

Description

RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 11 / 832,367, filed Aug. 1, 2007, which claims the benefit under 35 U.S.C. 119(e) of U.S. Provisional Application Ser. No. 60 / 821,078, filed Aug. 1, 2006, each of which is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The disclosed compositions and methods relate to molecular biology, more particularly to methods and compositions for nucleic acid isolation from a mixture, such as a sample, by using a nucleic acid oligomer that hybridizes nonspecifically to a target nucleic acid to separate it from other components of the mixture.BACKGROUND OF THE INVENTION[0003]Many molecular biology procedures such as in vitro amplification and in vitro hybridization of nucleic acids include some preparation of nucleic acids to make them effective in the subsequent procedure. Methods of nucleic acid purification may isolate all nucleic acids present in a sample, isolate different types of nucleic ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/68C12Q1/70
CPCC12Q1/6806C12Q1/6834C12Q1/70C12Q2565/519C12Q2565/518C12N15/1006C07H21/02C07H21/04C12N15/11C12N2310/18C12N2310/31C12N2310/321C12N2310/351C12N2330/30
Inventor BECKER, MICHAEL M.MAJLESSI, MEHRDAD R.
Owner GEN PROBE INC
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