title: Heterogeneity of primer extension products in asymmetric PCR is due both to cleavage by a structure-specific exo/endonuclease activity of DNA polymerases and to premature stops.
abstract: In PCR, DNA polymerases from thermophilic bacteria catalyze the extension of primers annealed to templates as well as the structure-specific cleavage of the products of primer extension. Here we show that cleavage by Thermus aquaticus and Thermus thermophilus DNA polymerases can be precise and substantial: it occurs at the base of the stem-loop structure assumed by the single strand products of primer extension using as template a common genetic element, the promoter-operator of the Escherichia coli lactose operon, and may involve up to 30% of the products. The cleavage is independent of primer, template, and triphosphates, is dependent on substrate length and temperature, requires free ends and Mg2+, and is absent in DNA polymerases lacking the 5'-->3' exonuclease, such as the Stoffel fragment and the T7 DNA polymerase. Heterogeneity of the extension products results also from premature detachment of the enzyme approaching the 5' end of the template.
authors.affiliation: Labortory of Molecular Genetics and Informatics, The Rockfeller University, New York, NY 10021-6399, USA.
authors.forename: G
authors.initials: G
authors.lastname: Tombline
authors.forename: D
authors.initials: D
authors.lastname: Bellizzi
authors.forename: V
authors.initials: V
authors.lastname: Sgaramella
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004706:Endodeoxyribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D013379:Substrate Specificity; D019914:Taq Polymerase; D013698:Templates, Genetic; D013824:Thermus / Q000201:enzymology*; D016963:Thermus thermophilus / Q000201:enzymology*
chemical_list: D017931:DNA Primers; D019914:Taq Polymerase; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases