LENGTH AND BASE COMPOSITION OF PCR-AMPLIFIED NUCLEIC-ACIDS USING MASSMEASUREMENTS FROM ELECTROSPRAY-IONIZATION MASS-SPECTROMETRY

A generally applicable algorithm has been developed to allow base comp osition of polymerase chain reaction (PCR) products to be determined h em mass spectrometrically measured molecular weights and the complemen tary nature of DNA. Mass measurements of arbitrary precision for singl e-stranded DNA species are compatible with an increasingly large numbe r of possible base compositions as molecular weight increases. For exa mple, the number of base compositions that are consistent with a molec ular weight of 35 000 is similar to 6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of st andard 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 uni quely defined at 0.001% mass precision. Taking into account the low mi sincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known sequence (generally 1 5-20-mer in size, which flank the targeted region), this reduces the n umber of possible base combinations to only similar to 3 at MW = 35 00 0. In addition, the number of base pairs (i.e., length of the DNA mole cule) is uniquely defined. We show that the use of modified bases in P CR or post-PCR modification chemistry allows unique solutions for the base composition of the PCR product with only modest mass measurement precision.