Method for analyzing the nucleotide sequence of a polynucleotide by oligonucleotide extension on an array

A method for determining the nucleotide sequence of a nucleic acid molecule which comprises the steps of:

• (A) providing an array of nested primer oligonucleotides on a solid support, each array position containing a different array member having a predetermined sequence;
• (B) incubating the oligonucleotides of said array in the presence of a preparation of said nucleic acid molecules a polymerase and at least one chain terminator nucleotide;
• wherein said incubation is under conditions sufficient to permit DNA hybridization to occur between the oligonucleotides of said incubation and said nucleic acid molecule;
• (C)
• (1) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is hybridized to said nucleic acid molecule, permitting the oligonucleotide hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, wherein for each hybridized oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that oligonucleotide's 3′ terminal nucleotide residue;
• then performing step (D);
• (2) in the case wherein the 3′ terminal nucleotide of an oligonucleotide is not hybridized to said nucleic acid molecule, either:
• (a) not permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized oligonucleotide, or
• (b) permitting the removal of any non-hybridized nucleotide residues from the 3′ terminus of said hybridized oligonucloetides, so as to form a truncated primer oligonucleotide whose 3′ terminus is hybridized to said nucleic acid molecule, and then permitting polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3′ terminus of said hybridized truncated oligonucleotide, wherein for each hybridized truncated oligonucleotide being so extended, said incorporated nucleotide residue is complementary to the nucleotide residue immediately 5′ to the nucleotide residue of the nucleic acid molecule that is hybridized with that truncated oligonucleotide's 3′ terminal nucleotide residue;
• then performing step (D);
• (D) determining, at each array position at which an oligonucleotide has incorporated a single chain terminator nucleotide residue, the identity of the incorporated chain terminator nucleotide residue;
• and
• (E) determining the nucleotide sequence of said nucleic acid molecule from the determined identity of the incorporated nucleotide of primer oligonucleotides of said array, and known sequence of the oligonucleotide at each array position.