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Method of mapping restriction sites in polynucleotides

  • US 6,518,023 B1
  • Filed: 03/27/2000
  • Issued: 02/11/2003
  • Est. Priority Date: 06/27/1997
  • Status: Expired due to Term
First Claim
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1. A method of ordering restriction fragments of a target polynucleotide, the method comprising the steps of:

  • (a) (i) producing a first population of restriction fragments by digestion of the target polynucleotide with a first restriction endonuclease having a first recognition site, (ii) attaching each restriction fragment of the first population to a solid phase support by one end, such that copies of each restriction fragment are attached to spatially discrete regions of one or more solid phase supports;

    (iii) determining the nucleotide sequence of a portion of each free end of each restriction fragment of the first population;

    (iv) digesting each restriction fragment of the first population with a second restriction endonuclease to form a first set of truncated support-bound restriction fragments, the second restriction endonuclease having a second recognition site different from that of the first restriction endonuclease, wherein at least one of said restriction endonucleases has a 4-basepair recognition sequence;

    (v) determining the nucleotide sequence of a portion of each free end of each truncated restriction fragment of the first set, so that an ordered pair of sequences is obtained for each restriction fragment of the first population;

    (b) (i) producing a second population of restriction fragments by digestion of the target polynucleotide with the second restriction endonuclease, (ii) attaching each restriction fragment of the second population to a solid phase support by one end, such that copies of each restriction fragment are attached to spatially discrete regions of one or more solid phase supports;

    (iii) determining the nucleotide sequence of a portion of each free end of each restriction fragment of the second population;

    (iv) digesting each restriction fragment of the second population with the first restriction endonuclease to form a second set of truncated support-bound restriction fragments;

    (v) determining the nucleotide sequence of a portion of each free end of each truncated restriction fragment of the second set, so that an ordered pair of sequences is obtained for each restriction fragment of the second population; and

    (c) ordering the restriction fragments produced by the first and second restriction endonucleases by aligning the matching nucleotide sequences from the ordered pairs of sequences from the first and second populations of restriction fragments;

    wherein each said step of attaching includes;

    attaching an oligonucleotide tag from a repertoire of tags to each restriction fragment, such that each oligonucleotide tag from the repertoire is selected from the same minimally cross-hybridizing set of oligonucleotides;

    wherein each of said oligonucleotide tags differs from every other oligonucleotide tag of said minimally cross-hybridizing set by at least three nucleotides;

    sampling said first population of restriction fragments such that substantially all different restriction fragments in said first population have different oligonucleotide tags attached; and

    specifically hybridizing the oligonucleotide tags with their respective tag complements, which are attached to spatially discrete regions on one or more solid phase supports;

    and wherein each step of determining includes;

    ligating to the free end of each support-bound restriction fragment, an encoded adaptor having a protruding strand which forms a perfectly matched duplex with said free end, said encoded adaptor further comprising an oligonucleotide tag selected from a minimally cross-hybridizing set of oligonucleotides, and a nuclease recognition site of a nuclease whose cleavage site is separate from its recognition site;

    specifically hybridizing a labeled tag complement to said oligonucleotide tag of the encoded adaptor, identifying the type and sequence of nucleotides in the free end of the restriction fragment in accordance with the label carried by the tag complement;

    cleaving the fragment with a nuclease recognizing the nuclease recognition site of the encoded adaptor, such that the fragment is shortened by one or more nucleotides; and

    repeating said ligating, hybridizing of labeled tag complements, and identifying steps, until a desired length of the nucleotide sequence of the end of the fragment is determined.

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