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Method for non-redundant library construction

  • US 6,727,068 B2
  • Filed: 01/23/2002
  • Issued: 04/27/2004
  • Est. Priority Date: 01/24/2001
  • Status: Expired due to Fees
First Claim
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1. A method for constructing a nucleic acid library comprising:

  • a) obtaining a population of end-labeled double-stranded cDNA molecules;

    b) dividing said population into a first portion and a second portion;

    c) digesting said first portion with at least one sequence-specific endonuclease wherein said first portion is divided into one pool per endonuclease;

    d) digesting said second portion with at least one (Y) endonuclease having a degenerate recognition sequence and fractionating the digested cDNA into YXz pools where Y is the number of degenerate endonucleases, X is the the extent of degeneracy and z is the number of bases;

    e) isolating nucleic acid fragments from said endonuclease digestions of said first and second portions using said end-labels;

    f) digesting the fragments of said first portion with said at least one endonuclease of d) sequence and fractionating the digested cDNA into YXz pools where Y is the number of degenerate endonucleases, X is the the extent of degeneracy and z is the number of bases;

    g) digesting the fragments of said second portion with said at least one endonuclease of c) wherein the second portion pools are divided into one pool per endonuclease;

    h) removing labeled nucleic acid fragments from said first and second portions while retaining unlabeled fragments;

    i) adding a population of adapters to each of the pools, said population containing adapters specific for the endonucleases used, wherein each adapter comprises a first region specific to a particular endonuclease used and a second region containing a primer binding site;

    j) hybridizing and ligating said adapters to said unlabeled fragments;

    k) amplifying the fragments using the adapters; and

    l) separating the amplified fragments by size.

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