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Methods for polynucleotide synthesis and articles for polynucleotide hybridization

  • US 6,083,726 A
  • Filed: 02/03/1998
  • Issued: 07/04/2000
  • Est. Priority Date: 02/03/1998
  • Status: Expired due to Term
First Claim
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1. A method for synthesizing a polynucleotide comprising the following steps:

  • a) obtaining a double-stranded polynucleotide wherein one or more nucleotides at one end of one strand (α

    ) extend as a single-stranded structure beyond the end of the other (β

    ) strand to form a sticky end;

    b) obtaining a third polynucleotide strand (γ

    ) which has a nucleotide sequence at one end which can hybridize to the sticky end of the α

    polynucleotide, wherein the bases of a number p of nucleotides in the γ

    strand adjacent to the terminal nucleotide sequence complementary to the sticky end of the α

    strand are universal bases, where p is a number of new nucleotides to be added to the end of the α

    strand,hybridizing the γ

    strand to said sticky end, andligating the hybridized end of the γ

    strand to the end of the β

    strand;

    c) obtaining a fourth polynucleotide strand (ε

    ) which is complementary to the single-stranded portion of the ligated γ

    strand, wherein the ε

    strand has p nucleotides at its end which are to be added to the α

    strand, which nucleotides pair with the p universal bases of the γ

    strand andhybridizing the ε

    strand to the γ

    strand, andligating the end of the hybridized ε

    strand to the adjacent end of the α

    strand;

    d) cutting the resulting double-stranded DNA complexes with a restriction enzyme so that the end of the α

    strand has p bases more than it had in step a, and so that one or more nucleotides of the lengthened end of the α

    strand extend beyond the end of the β

    strand to form a sticky end; and

    e) repeating steps b-d until the newly synthesized polynucleotide has the desired nucleotide sequence.

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