DNA amplification and subtraction techniques

  • US 6,107,023 A
  • Filed: 06/17/1988
  • Issued: 08/22/2000
  • Est. Priority Date: 06/17/1988
  • Status: Expired due to Term
First Claim
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1. A method of isolating duplex DNA fragments which are present in a mixture of different-sequence duplex DNA fragments derived from a positive source, but absent from a mixture of different-sequence duplex DNA fragments derived from a negative source, said method comprisingattaching a double-strand linker to the positive-source fragments, and separately, to the negative-source fragments, by ligating the linker to both strands of said positive-source and negative-source fragments, at both ends of said positive-source and negative-source fragments,amplifying the number of each linker-carrying fragment in each fragment mixture by successively repeating the steps of (i) denaturing the fragments to produce single fragment strands with linker regions at each strand end, (ii) hybridizing the single strands with a single-strand primer whose sequence is complementary to the linker region at one end of each strand, to form a strand/primer complex, and (iii) converting the strand/primer complexes to double-strand fragments in the presence of polymerase and deoxynucleotides,denaturing the amplified fragments in the two amplified fragment mixtures and hybridizing the denatured fragments in the two mixtures under conditions in which the linker regions associated with the positive-source strands do not hybridize with the linker regions associated with the negative-source strands, andselectively isolating DNA species which are not hybridized with DNA fragment strands from the negative source.

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