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Alternative dye-labeled primers for automated DNA sequencing

  • US 5,614,386 A
  • Filed: 06/23/1995
  • Issued: 03/25/1997
  • Est. Priority Date: 06/23/1995
  • Status: Expired due to Fees
First Claim
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1. A method for distinguishing polynucleotide sequences in a hybridization method of DNA sequencing, said method comprising the steps of:

  • synthesizing a first, a second, a third and a fourth class of oligonucleotides, wherein all of said classes of oligonucleotides have a same length, said first, second, third and fourth classes of oligonucleotides differ from the oligonucleotides of each other class by one nucleotide base at a 3'"'"', a 5'"'"' or an internal position, and each oligonucleotide of the first class has a dideoxyadenosine at said position and is labeled with a first fluorophore;

    each oligonucleotide in the second class has a dideoxycytidine at said position and is labeled with a second fluorophore;

    each oligonucleotide in the third class having a dideoxyguanosine at said position and is labeled with a third fluorophore; and

    each oligonucleotide in the fourth class has a dideoxythymidine at said position and is labeled with a fourth fluorophore;

    wherein at least one of said fluorophore is a 4,4-difluoro-4-bora-3A,4A-diaza-s-indacene (BODIPY®

    ) fluorophore;

    hybridizing said oligonucleotides to a single-stranded DNA target immobilized to a solid support, wherein said solid support is in a grid format, to form a hybridized product;

    washing said hybridized product to remove any unhybridized oligonucleotide or target;

    illuminating with an illumination beam the solid support, said illumination beam being capable of causing said BODIPY®

    fluorophores to fluoresce; and

    identifying the classes of polynucleotides in the bands by the fluorescence or absorption spectrum of the dyes.

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