CPC C12Q 1/6869 (2013.01) [C12Q 1/6834 (2013.01)] | 30 Claims |
1. A method of identifying a detectable nucleotide analogue, said method comprising:
a) hybridizing a first sequencing primer to a polynucleotide immobilized to a solid support, wherein said polynucleotide comprises a primer binding sequence and a template nucleic acid sequence comprising a plurality of cleavable sites;
b) contacting said first sequencing primer hybridized to said polynucleotide with a first detectable nucleotide analogue and a polymerase to form a first polymerase complex, detecting the first polymerase complex, and identifying said first detectable nucleotide analogue;
c) incorporating one or more nucleotides into said first sequencing primer hybridized to said polynucleotide with a polymerase to create an extension strand hybridized to said polynucleotide;
d) cleaving the at least one cleavable site, thereby generating a polynucleotide fragment immobilized to the solid support and at least one non-immobilized polynucleotide fragment, wherein said polynucleotide fragment immobilized to the solid support is hybridized to said extension strand;
e) removing the one or more non-immobilized polynucleotide fragments; and
f) hybridizing a second sequencing primer to said extension strand and contacting said second sequencing primer hybridized to said extension strand with a second detectable nucleotide analogue and a polymerase to form a second polymerase complex, detecting the second polymerase complex, and identifying said second detectable nucleotide analogue.
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