CPC C12Q 1/6869 (2013.01) [G01N 27/44717 (2013.01); G01N 27/44791 (2013.01); G01N 33/48721 (2013.01)] | 14 Claims |
1. A method comprising:
(i) preparing a nucleic acid construct by ligating a polynucleotide bridging moiety to a 3′ terminus of a first strand and a 5′ terminus of a second strand of a double stranded nucleic acid, wherein the first strand of the double stranded nucleic acid comprises a first nucleic acid sequence and the second strand of the double stranded nucleic acid comprises a second nucleic acid sequence that is complementary to the first nucleic acid sequence;
(ii) using a single molecule sequencing apparatus to perform a sequencing reaction that reads through the first strand, the bridging moiety, and the second strand to produce orthogonal proof-reading sequence information for a methylated nucleobase in the first strand of the construct, wherein the movement of the nucleic acid construct through the single molecule sequencing apparatus is controlled by a Phi29 DNA polymerase;
(iii) comparing the sequence information between the first nucleic acid sequence and the second nucleic acid sequence; and
(iv) determining the presence of a methylated nucleobase in the first sequence.
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