Length and base composition of PCR-amplified nucleic acids using mass measurements from electrospray ionization mass...

A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35,000 is approximately 6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 15-20-mer in size, which flank the targeted region), this reduces the number of possible base combinations to only approximately 3 at MW = 35,000. In addition, the number of base pairs (i.e., length of the DNA molecule) is uniquely defined. We show that the use of modified bases in PCR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.