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Electrophoretic detection and separation of mutant DNA using replaceable polymer matrices

  • US 5,633,129 A
  • Filed: 02/03/1994
  • Issued: 05/27/1997
  • Est. Priority Date: 07/13/1989
  • Status: Expired due to Fees
First Claim
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1. A method of resolving mutant DNA from non-mutant DNA in a DNA sample which, as obtained or modified, contains double stranded mutant DNA and double stranded non-mutant DNA, each with two iso-melting domains, a first iso-melting domain referred to as a low temperature iso-melting domain, which melts at a first temperature and a second iso-melting domain, referred to as a high temperature iso-melting domain, which melts at a second, higher temperature, wherein the mutant DNA contains a mutation of interest in the low temperature iso-melting domain, comprising the steps of:

  • a) isolating the DNA sample from a biological source;

    b) fragmenting DNA in the DNA sample isolated in a) to obtain fragmented double-stranded DNA of interest;

    c) melting and reannealing the fragmented double-stranded DNA of interest obtained in step b) under conditions appropriate to form duplexed DNA, thereby producing a mixture of DNA heteroduplexes and DNA homoduplexes;

    d) introducing the mixture produced in step c) into a replaceable polymer matrix contained within a capillary column, thereby producing a replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes;

    e) subjecting the replaceable polymer matrix containing the mixture of DNA heteroduplexes and DNA homoduplexes to a high electrical field and partially denaturing conditions, wherein the partially denaturing conditions are constant denaturing conditions, whereby the DNA heteroduplexes partially melt and the DNA homoduplexes do not melt and the DNA heteroduplexes migrate at a slower velocity in the replaceable polymer matrix then the DNA homoduplexes, resulting in the separation of DNA heteroduplexes from DNA homoduplexes.

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