Polymerase chain reaction amplification and sequence analysis of human mutant adenine phosphoribosyltransferase genes: the nature and frequency of errors caused by Taq DNA polymerase.
Using the polymerase chain reaction (PCR) with Taq DNA polymerase, we have amplified a 2.4-kb fragment of genomic DNA containing the adenine phosphoribosyltransferase (APRT) gene from patients with APRT deficiency. Several clones from each patient were sequenced after subcloning the PCR product into M13mp18. Selected regions of the amplified fragment were also sequenced directly. This enabled us to distinguish PCR-induced errors from endogenous mutations and polymorphisms in each clone. 44 PCR errors were found in a total of 57,94 kb of DNA sequenced from 25 clones from 7 patients. All the errors were due to the PCR process and not to subcloning, as shown by sequence analysis of 5 APRT-positive clones isolated from a phage genomic library.
Chen
J
J
Department of Medical Genetics, Indiana University School of Medicine, Indianapolis 46202.
Sahota
A
A
Stambrook
P J
PJ
Tischfield
J A
JA
D000228:Adenine Phosphoribosyltransferase / Q000172:deficiency / Q000235:genetics*; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics; D004259:DNA-Directed DNA Polymerase; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011237:Predictive Value of Tests; D019914:Taq Polymerase
D004277:DNA, Single-Stranded; D004247:DNA; D000228:Adenine Phosphoribosyltransferase; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase