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MULTISIGNAL LABELING REAGENTS AND PROCESSES AND USES THEREFOR

  • US 20180127455A1
  • Filed: 12/21/2017
  • Published: 05/10/2018
  • Est. Priority Date: 04/03/2003
  • Status: Active Grant
First Claim
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1. A method of producing a multisignal labeling reagent, the method comprising(a) obtaining(i) a primer comprising an oligonucleotide and a first reactive group or a first partner of a first binding pair at the 5′

  • end of the oligonucleotide;

    (ii) a template comprising a nucleic acid comprising a first sequence that is complementary to the oligonucleotide and a second sequence that extends in the 5′

    direction from the first sequence;

    (iii) a polymerase capable of extending the oligonucleotide along the template nucleic acid when the template nucleic acid is hybridized to the oligonucleotide at the first sequence; and

    (iv) nucleotide triphosphates (NTPs) or analogs thereof that are capable of being incorporated into the extended oligonucleotide, wherein at least one of the NTPs or analogs comprises a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair; and

    (b) combining the primer, template, polymerase and NTPs or analogs under conditions such that the oligonucleotide hybridizes to the first sequence and is extended along the second sequence, where the extended oligonucleotide comprises at least two NTPs or analogs incorporated therein that comprise a non-radioactive detectable label, a second reactive group or a first partner of a second binding pair;

    wherein,(A) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a second reactive group, the method further comprises combining the extended oligonucleotide with a first compound comprising a non-radioactive detectable label covalently linked to a moiety capable of reacting with the second reactive group such that the label is covalently linked to the extended primer, and(B) if at least one of the two or more NTPs or analogs incorporated into the extended oligonucleotide comprises a first partner of the second binding pair, the method further comprises combining the extended primer with a second compound comprising the non- radioactive detectable label covalently linked to a second binding partner of the second binding pair.

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