Replica amplification of nucleic acid arrays

A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of:

• a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;
• b) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′ end of the primer if it is complementary to the next adjacent base in the sequence to be determined;
• c) detecting incorporated label by monitoring fluorescence;
• d) repeating steps (b)-(c) with each of the remaining three labeled deoxynucleoside triphosphates in turn;
• e) photobleaching said array;
• and
• f) repeating steps (b)-(e) until the nucleotide sequence is determined.

A method of determining the nucleotide sequence of the features of an array of immobilized nucleic acids comprising the steps of:

• a) adding a mixture comprising an oligonucleotide primer and a template-dependent polymerase to an array of immobilized nucleic acid features under conditions permitting hybridization of the primer to the immobilized nucleic acids;
• b) adding a first mixture of three unlabeled deoxynucleoside triphosphates under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;
• c) adding a second mixture of three unlabeled deoxynucleoside triphosphates, said second mixture comprising the deoxynucleoside triphosphate not included in the mixture of step (b), under conditions which permit incorporation of deoxynucleotides to the end of the primer if they are complementary to the next adjacent base in the sequence to be determined;
• d) repeating steps (b)-(c) for a predetermined number of cycles;
• e) adding a single, fluorescently labeled deoxynucleoside triphosphate to the mixture under conditions which permit incorporation of the labeled deoxynucleotide onto the 3′ terminus of the primer if it is complementary to the next adjacent base in the sequence to be determined;
• f) detecting incorporated label by monitoring fluorescence;
• g) repeating steps (e)-(f), with each of the remaining three labeled deoxynucleoside triphosphates in turn;
• h) photobleaching said array;
• and
• i) repeating steps (e)-(h) until the nucleotide sequence is determined.