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Methods of synthesizing labeled polynucleotides by ligation of multiple oligomers

  • US 6,001,614 A
  • Filed: 02/02/1999
  • Issued: 12/14/1999
  • Est. Priority Date: 07/24/1998
  • Status: Expired due to Term
First Claim
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1. A method comprising:

  • a) providing a reaction mixture comprising a single stranded nucleic acid template, a primer having at least 15 bases which is complementary to a portion of the single stranded nucleic acid template and a plurality of oligonucleotide 5'"'"'-monophosphates wherein each oligonucleotide 5'"'"'-monophosphate consists of not more than 10 bases and wherein each of the oligonucleotide 5'"'"'-monophosphates is labeled;

    b) hybridizing the primer with the template under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to form a primer-template hybrid having a single stranded region and a double stranded region;

    c) ligating more than one of the plurality of labeled oligonucleotide 5'"'"'-monophosphates in a contiguous manner onto the primer in one continuous process under conditions which permit stable hybridization of the primer but not stable hybridization of the oligonucleotide 5'"'"'-monophosphates to extend the double stranded region and synthesize a labeled complementary nucleic acid strand, wherein ligation of oligonucleotide 5'"'"'-monophosphates only occurs in the presence of the hybridized primer.

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