Method for characterizing nucleic acid molecules

A method for characterizing a nucleic acid molecule comprising:

• a) synthesizing DNA in the presence of a reaction mixture comprising:
• (i) a nucleic acid template,
• (ii) a primer molecule that contains a nucleotide sequence at least part of which is sufficiently complementary to a portion of the template to hybridize therewith,
• (iii) an enzyme that extends the primer so that a DNA molecule may be synthesized,
• (iv) four canonical deoxynucleoside triphosphates, and
• (v) at least one non-canonical deoxynucleoside triphosphate, wherein the non-canonical deoxynucleoside triphosphate is one that is incorporated with fidelity into the synthesized DNA in place of only one canonical deoxynucleoside triphosphate;
• b) contacting the synthesized DNA with an N-glycosylase that excises a base portion of the non-canonical deoxynucleoside triphosphate from said synthesized DNA, whereby an abasic site is created;
• c) treating the DNA in such manner that said treatment results in breakage of a phosphodiester backbone at all abasic sites, whereby at least two DNA fragments are created;
• and
• d) separating the fragments according to size.