FACTORS AFFECTING FIDELITY OF DNA-SYNTHESIS DURING PCR AMPLIFICATION OF D(C-A)(N)CENTER-DOT-D(G-T)(N) MICROSATELLITE REPEATS

The susceptibility of microsatellite DNA sequences to insertions and d eletions in vivo makes them useful for genetic mapping and for detecti ng genomic instability in tumors, An in vitro manifestation of this in stability is the production of undesirable frameshift products during amplification of (dC-dA)(n) .(dG-dT)(n) microsatellites in the polymer ase chain reaction (PCR), These products differ from the primary produ ct by multiples of 2 nucleotides, We have tested the hypothesis that f actors known to affect the fidelity of DNA synthesis may affect (dC-dA ), (dG-dT), frameshifting during the PCR, Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the us e of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products dur ing amplification. However, 3'-->5' exonuclease activity in thermophil ic DNA polymerases inhibited the accumulation of PCR products containi ng nontemplated 3' terminal nucleotides, Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolab ile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonucle ase-deficient Klenow fragment greatly decreased the production of fram eshift products, This method can improve the resolution of heterozygou s or mutant (dC-dA), (dG-dT), alleles differing in size by one or two repeat units.