Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats.
The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors. An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR). These products differ from the primary product by multiples of 2 nucleotides. We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR. Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification. However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides. Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products. This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units.
Hite
J M
JM
Department of Pathology, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, PA 17033, USA.
Eckert
K A
KA
Cheng
K C
KC
D001483:Base Sequence; D004247:DNA / Q000138:chemical synthesis*; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D018900:Dinucleotide Repeats; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D018895:Microsatellite Repeats; D008969:Molecular Sequence Data; D011089:Polydeoxyribonucleotides / Q000138:chemical synthesis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013696:Temperature; D013698:Templates, Genetic
D011089:Polydeoxyribonucleotides; C044165:poly(d(G-T).d(C-A)); D004247:DNA; C088943:UlTma DNA polymerase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V