Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the blaTEM gene

Yaroslav K Masalov; Rustam N Heydarov; Igor A Shashkov; Igor V Chebotar; Alexander S Zasedatelev; Nikolay A Mayansky; Vladimir M Mikhailovich

doi:10.1016/j.mimet.2019.03.018

Exogenous contaminating DNA in Taq polymerases: A method to avoid false-positive results when detecting the bla_TEM gene

J Microbiol Methods. 2019 May:160:36-41. doi: 10.1016/j.mimet.2019.03.018. Epub 2019 Mar 20.

Authors

Yaroslav K Masalov¹, Rustam N Heydarov¹, Igor A Shashkov¹, Igor V Chebotar², Alexander S Zasedatelev¹, Nikolay A Mayansky², Vladimir M Mikhailovich³

Affiliations

¹ Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119991, Russia.
² National Medical Research Center for Children's Health, Ministry of Health, 2 Lomonosovsky Pros., Moscow 119991, Russia.
³ Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 32 Vavilova Str., Moscow 119991, Russia. Electronic address: v.mikhailovich@gmail.com.

PMID: 30904556
DOI: 10.1016/j.mimet.2019.03.018

Abstract

In the course of developing an assay to identify genes responsible for antibiotic resistance in gram-negative bacteria, it has been found that standard (not DNA-free) Taq DNA polymerases were contaminated with bla_TEM gene fragments that varied in length and quantities. The complete bla_TEM gene sequence was either absent or was detected in infinitesimal amounts. We developed an approach to avoid false-positive findings caused by contaminating bla_TEM gene sequences in conventional polymerases. The method is based on selection of a target sequence to be detected within the bla_TEM gene in such a way that the chosen sequence is amplified with primers incapable of amplifying contaminating DNA sequences of the polymerase.

Keywords: Contaminating DNA; False-positive results; Microarray; Taq DNA polymerase; bla(TEM) gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

DNA Contamination*
DNA Primers / chemistry
DNA, Bacterial / genetics*
Escherichia coli / genetics
False Positive Reactions
Polymerase Chain Reaction / methods*
Taq Polymerase / analysis*

Substances

DNA Primers
DNA, Bacterial
Taq Polymerase