Method of determining the nucleotide sequence of oligonucleotides and dna molecules

A method of DNA sequencing comprising the steps of:

• (a) providing a template system comprising at least one nucleic acid molecule of unknown sequence hybridized to a primer oligonucleotide in the presence of a exonuclease deficient DNA polymerase;
• (b) contacting the template system with a single type of deoxyribonucleotide under conditions which allow extension of the primer by incorporation of at least one deoxyribonucleotide to the 3′ end of the primer to form an extended primer;
• (c) detecting whether extension of the primer has occurred thereby identifying the deoxyribonucleotide added to the 3′ end of the primer;
• (d) detecting the number of deoxyribonucleotides incorporated into the primer;
• (e) removing unincorporated deoxyribonucleotide;
• (f) contacting the template system with a mixture including an exonuclease proficient DNA polymerase, an exonuclease deficient DNA polymerase and the identified deoxyribonucleotide of step (b);
• (g) removing the mixture of step (f);
• and
• (h) repeating steps (a) through (g) to determine the nucleotide sequence of the nucleic acid molecule.

A method for discriminating between the in-phase and out-of-phase sequencing signals comprising:

• (a) detecting and measuring error signals thereby determining the size of the trailing strand population;
• (b) between the 3′ terminus of the trailing strand primers and the 3′ terminus of the leading strand primers;
• (c) simulating the occurrence of an extension failure at a point upstream from the 3′ terminus of the leading strands thereby predicting at each extension step the exact point in the sequence previously traversed by the leading strands to which the 3′ termini of the trailing strands have been extended;
• (d) predicting for each dNTP introduced the signal to be expected from correct extension of the trailing strands;
• and
• (e) subtracting the predicted signal from the measured signal to yield a signal due only to correct extension of the leading strand population.