Title: DNA purification by triplex-affinity capture and affinity capture electrophoresis

Abstract

The invention provides a method for purifying or isolating double stranded DNA intact using triple helix formation. The method includes the steps of complexing an oligonucleotide and double stranded DNA to generate a triple helix and immobilization of the triple helix on a solid phase by means of a molecular recognition system such as avidin/biotin. The purified DNA is then recovered intact by treating the solid phase with a reagent that breaks the bonds between the oligonucleotide and the intact double stranded DNA while not affecting the Watson-Crick base pairs of the double helix. The present invention also provides a method for purifying or isolating double stranded DNA intact by complexing the double stranded DNA with a specific binding partner and recovering the complex during electrophoresis by immobilizing it on a solid phase trap imbedded in an electrophoretic gel.

Inventors:

Cantor, Charles R ^[1]; Ito, Takashi ^[2]; Smith, Cassandra L ^[1]

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+ Show Inventor Affiliations

1. Boston, MA
2. Chiba, JP

Issue Date:

1996-01-01

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

OSTI Identifier:

870243

Patent Number(s):

5482836

Assignee:

Regents of University of California (Oakland, CA)

Patent Classifications (CPCs):

C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

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C - CHEMISTRY C12 - BIOCHEMISTRY C12N - MICROORGANISMS OR ENZYMES

C12N15/1003 - {Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods
C12N15/101 - {by chromatography, e.g. electrophoresis, ion-exchange, reverse phase}

C - CHEMISTRY C12 - BIOCHEMISTRY C12Q - MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS

C12Q1/6806 - Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
C12Q1/6839 - Triple helix formation or other higher order conformations in hybridisation assays
C12Q2527/119 - pH
C12Q2537/119 - Triple helix formation

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DOE Contract Number:  

AC03-76SF00098

Resource Type:

Patent

Country of Publication:

United States

Language:

English

Subject:

dna; purification; triplex-affinity; capture; affinity; electrophoresis; provides; method; purifying; isolating; double; stranded; intact; triple; helix; formation; steps; complexing; oligonucleotide; generate; immobilization; solid; phase; means; molecular; recognition; avidin; biotin; purified; recovered; treating; reagent; breaks; bonds; affecting; watson-crick; base; pairs; specific; binding; partner; recovering; complex; immobilizing; trap; imbedded; electrophoretic; triple helix; stranded dna; binding partner; solid phase; double stranded; affinity capture; double helix; base pairs; base pair; specific binding; /435/204/999/