CPC C12Q 1/6806 (2013.01) [C12Q 1/6809 (2013.01)] | 4 Claims |
1. A method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising:
(a) contacting a target nucleic acid molecule comprising the known target nucleotide sequence with an initial target-specific primer under hybridization conditions;
(b) performing a template-dependent extension reaction that is primed by a hybridized initial target-specific primer and that uses the target nucleic acid molecule as a template;
(c) contacting an extension product of step (b) with a plurality of tailed random primers under hybridization conditions;
(d) performing a template-dependent extension reaction that is primed by a hybridized tailed random primer and that uses the extension product of step (b) as a template;
(e) amplifying a portion of an extension product of step (d) with a first tail primer and a first target-specific primer; and
(f) sequencing an amplification product from step (e) using a first or second sequencing primer;
wherein each of the plurality of tailed random primers comprises (i) a common sequence and (ii) a 3′-end comprising about 3 to about 15 random nucleotides;
wherein the initial target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid;
wherein the first target-specific primer comprises a 3′ portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence and is nested with respect to the initial target-specific primer; and
wherein the first tail primer comprises a nucleic acid sequence identical to a portion of the common sequence.
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