Elimination of Bacterial DNA from Taq DNA Polymerases by Restriction Endonuclease Digestion

Abstract

The incidence of false positives due to the presence of bacterial DNA in Taq DNA polymerase is an obstacle to the use of PCR in the diagnosis of infection. We describe a method that uses a restriction enzyme to destroy the ability of contaminating sequences to act as templates for a nested PCR which uses primers based on the 16S rRNA genes. The method was used prior to a PCR that amplified 10 fg of bacterial DNA. This method can be readily adapted to suit other sensitive PCRs required for clinical applications.

Reagent contamination can critically impact sequence-based microbiome analyses

“…A number of methods have been devised to treat reagents in order to reduce potential contamination, including: gamma 45 or UV radiation 13,46–48 ; DNase treatment 10,13,47,49–51 restriction digests 10,13,47,52,53 caesium chloride density gradient centrifugation 10 , and DNA intercalation and crosslinking with 8-methoxypsoralen 47,54 propidium monoazide 55 or ethidium monoazide 56,57 . However tests of these methods show varying levels of success.…”

Investigation of bacterial and viral agents and immune status in Behcet’s disease patients from Iran

“…Even today there is no procedure to eliminate this impurity, even though different methods have been tested to improve the detection limit. For example, restriction enzymes like SAU 3A1, 28 Alu1 29 or HaeIII 16 were used to reduce the level of contamination but results were inconsistent. In our study we tried to improve the sensitivity also using SAU 3A1 cutting short DNA fragments, but we were not able to improve the sensitivity.…”

PCR-based diagnostics for infectious diseases: uses, limitations, and future applications in acute-care settings

“…Attempts to decontaminate PCR materials have involved nearly all known methods of destroying DNA including ultraviolet irradiation, chemical treatment, and enzymatic digestion. 107,108 None of these methods has been shown to be entirely effective without significant diminution of assay sensitivity. We have recently reported an alternative method that uses a size-based ultrafiltration step for reducing contaminating DNA from PCR reagents, primers, and DNA polymerase before amplification.…”