Alternative dye-labeled primers for automated DNA sequencing

Claim 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.

Claim 2

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.