Replica amplification of nucleic acid arrays

A method for determining the nucleotide sequence of the features of an immobilized nucleic acid array, said method comprising the steps of:

• a) ligating a first double-stranded nucleic acid probe to one end of a nucleic acid of a feature of said array, said first double stranded nucleic acid probe having a restriction endonuclease recognition site for a restriction endonuclease whose cleavage site is separate from its recognition site and which generates a protruding strand upon cleavage;
• b) identifying one or more nucleotides at the end of said polynucleotide by the identity of the first double stranded nucleic acid probe ligated thereto or by extending a strand of the polynucleotide or probe;
• c) amplifying the features of said array using a primer complementary to said first double stranded nucleic acid probe, such that only molecules which have been successfully ligated with said first double stranded nucleic acid probe are amplified to yield an amplified array;
• d) contacting said amplified array with support such that at least a subset of nucleic acid molecules produced by said amplifying are transferred to said support;
• e) covalently attaching said subset of nucleic acid molecules transferred in step (d) to said support to form a replica of said amplified array;
• f) cleaving the nucleic acid features of the array with a nuclease recognizing said nuclease recognition site of said probe such that the nucleic acid of the features is shortened by one or more nucleotides;
• and
• g) repeating steps (a)-(f) until the nucleotide sequences of the features of said array are 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 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;
• and
• e) repeating steps (b)-(d) 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;
• and
• h) repeating steps (e) -(g) until the nucleotide sequence is determined.

A method of determining the nucleotide sequence of the features of a micro-array of nucleic acid molecules, said method comprising the following steps:

• a) creating a micro-array of nucleic acid features in a linear arrangement within and along one side of a polyacrylamide gel, said gel further comprising one or more oligonucleotide primers, and a template-dependent polymerizing activity;
• b) amplifying the microarray of step (a);
• c) adding a mixture of deoxynucleoside triphosphates, said mixture comprising each of the four deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, said mixture further comprising chain-terminating analogs of each of the deoxynucleoside triphosphates dATP, dGTP, dCTP and dTTP, and said chain-terminating analogs each distinguishably labeled with a spectrally distinguishable fluorescent moiety;
• d) incubating said mixture with said micro-array under conditions permitting extension of said one or more oligonucleotide primers;
• e) electrophoretically separating the products of said extension within said polyacrylamide gel;
• and
• f) determining the nucleotide sequence of the features of said micro-array by detecting the fluorescence of the extended, terminated and separated reaction products within the gel.

A method of simultaneously amplifying a plurality of nucleic acids, said method comprising the steps of:

• a) creating a micro-array of immobilized oligonucleotide primers;
• b) incubating the microarray of step (a) with amplification template and a non-immobilized oligonucleotide primer under conditions allowing hybridization of said template with said oligonucleotide primers;
• c) incubating the hybridized primers and template of step (b) with a DNA polymerase activity, and deoxynucleotide triphosphates under conditions permitting extension of the primers;
• d) repeating steps (b) and (c) for a defined number of cycles to yield a plurality of amplified DNA molecules.