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.
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.
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Labortory of Molecular Genetics and Informatics, The Rockfeller University, New York, NY 10021-6399, USA.
Bellizzi
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Sgaramella
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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*
D017931:DNA Primers; D019914:Taq Polymerase; C068508:Tth polymerase; D004259:DNA-Directed DNA Polymerase; D004706:Endodeoxyribonucleases; D005090:Exodeoxyribonucleases