Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase.
Polymerase chain reaction (PCR) was performed with two polymerases. Thermus aquaticus DNA polymerase (Taq), and modified T7 DNA polymerase (Sequenase). Both polymerases were used to amplify the same portion of the human 18S rRNA gene. We report a PCR artifact, namely a deletion of 54 bp, when Taq polymerase was used to amplify a portion of the human 18S rRNA gene. PCR performed with Sequenase did not produce this artifact. The deletion eliminated a potentially stable hairpin loop. Our data are consistent with the following model for generation of the deletion: (i) the formation of an intrastrand hairpin, and (ii) polymerization across the base of the hairpin, thus deleting the nucleotide sequence in the hairpin. Furthermore, we show that the deletion occurs mainly during synthesis of the (-)DNA strand. Our observations suggest that similar artifacts may occur in other sequences containing stable secondary structures.
Cariello
N F
NF
Pathology Department, University of North Carolina, Chapel Hill 27599.
Thilly
W G
WG
Swenberg
J A
JA
Skopek
T R
TR
D001483:Base Sequence; D002872:Chromosome Deletion; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012337:RNA, Ribosomal, 18S / Q000235:genetics*; D013604:T-Phages / Q000201:enzymology; D019914:Taq Polymerase; D013824:Thermus / Q000201:enzymology
D012337:RNA, Ribosomal, 18S; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase