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Methods for generating target specific probes for solution based capture

  • US 8,986,958 B2
  • Filed: 03/30/2010
  • Issued: 03/24/2015
  • Est. Priority Date: 03/30/2009
  • Status: Active Grant
First Claim
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1. A method for generating a population of single-stranded nucleic acid probes, each probe comprising a predetermined nucleotide sequence, the method comprising:

  • a) providing a starting population of linear double-stranded nucleic acid precursor molecules each precursor molecule having (i) a probe region having the predetermined sequence which is flanked at a 5′ and

    a 3′

    end by a first and a second restriction enzyme recognition sequence for generating ligation substrates and for ligating a plurality of the double-stranded nucleic acid precursor molecules into head-to-tail concatemers (ii) the 5′

    flanking region including the first restriction enzyme recognition sequence and (iii) the 3′

    flanking region including the second restriction enzyme recognition sequence;

    b) contacting the 5′ and

    3′

    flanking regions of the linear double-stranded nucleic acid precursor molecules with a first and a second restriction enzyme to cleave the first and second restriction enzyme recognition sequences so as to generate ligation substrates;

    c) ligating the ligation substrates together so as to generate a plurality of random head-to-tail concatemers;

    d) amplifying the plurality of head-to-tail concatemers;

    e) contacting the amplified head-to-tail concatemers with the first and second restriction enzymes so as to release a plurality of double-stranded monomer linear precursor molecules; and

    f) selectively removing one strand of the double-stranded monomer linear precursor molecules so as to generate a population of single-stranded nucleic acid probes, each probe comprising the predetermined nucleotide sequence.

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