Patent ID: 7074597

Claim:
A method for determining the identity of a nucleotide present at a predetermined site in a DNA whose sequence immediately 3â€² of such predetermined site is known which comprises: (a) treating the DNA with an oligonucleotide primer whose sequence is complementary to such known sequence so that the oligonucleotide primer hybridizes to the DNA and forms a complex in which the 3â€² end of the oligonucleotide primer is located immediately adjacent to the predetermined site in the DNA; (b) simultaneously contacting the complex from step (a) with four different dideoxynucleotides each photocleavably attached to a label, in the presence of a polymerase under conditions permitting a labeled dideoxynucleotide to be added to the 3â€² end of the primer so as to generate a labeled single base extended primer, wherein each of the four different labeled dideoxynucleotides (i) is complementary to one of the four nucleotides present in the DNA and (ii) has a molecular weight which can be distinguished from the molecular weight of the other three labeled dideoxynucleotides using mass spectrometry; (c) (i) contacting the labeled single base extended primer with a solid surface coated with a compound that specifically interacts with the label attached to the dideoxynucleotide so as to thereby capture the extended primer on the surface, (ii) removing primers that have not been extended by a labeled dideoxynucleotide, and (iii) photocleaving the captured labeled single base extended primer so as to release it from the surface; and (d) determining the difference in molecular weight between the labeled single base extended primer and the oligonucleotide primer so as to identify the dideoxynucleotide incorporated into the single base extended primer and thereby determine the identity of the nucleotide present at the predetermined site in the DNA.