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.

FIG. 1

Nested PCRs using primer pairs 16F plus 16R and NR plus NF amplify a product in the absence of an added template. Lanes 1 to 5 were amplified by using Amplitaq LD (Perkin-Elmer, Cheshire, United Kingdom), lanes 6 to 10 were amplified with Amplitaq (Perkin-Elmer), and lanes 11 to 15 were amplified with Taq DNA polymerase (Stratagene, Amsterdam, The Netherlands). Lanes 1, 6, and 11 were positive controls for the outer PCRs; lanes 2, 7, and 12 were reagent controls for the outer reaction; lanes 3, 8, and 13 were positive controls for the nested reaction; lanes 4, 9, and 14 contained 1 μl of the first-round reagent control amplified with the nested primers; and lanes 5, 10, and 15 were reagent controls for the nested PCR. Lane 16 contained the molecular size marker (Promega, Wis.).

FIG. 2

Effect on product amplification of treating Taq DNA polymerase with decreasing amounts of Sau3A1. Positive reactions contained 20 ng of Escherichia coli NTCC 11151 DNA. Negative reaction mixtures contained no added template. (A) First round of amplification with primers 16F and 16R. Lanes 1, 3, 5, 7, 9, 11, and 13 were positive reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lanes 2, 4, 6, 8, 10, 12, and 14 were negative reaction mixtures treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lane 15 contained the molecular size marker (GIBCO, Paisley, Scotland). (B) Second round of amplification with primers NF and NR. Lanes 1 to 7 contained 1 μl of the negative reaction mixtures from round 1 that had been treated with 4, 2, 1, 0.5, 0.25, 0.13, and 0 U of Sau3AI, respectively. Lane 8 contained the positive control for the amplification, and lane 9 contained the reagent control. Lane 10 contained the molecular size marker (GIBCO).

FIG. 3

Sensitivity of nested PCR amplification of E. coli NTCC 11151 DNA using primer pairs 16F plus 16R and NF plus NR. The template was E. coli NTCC 11151 DNA purified as described in the text. Lanes 1 to 8 contained the products of the amplification of 10 ng, 1 ng, 100 pg, 10 pg, 1 pg, 100 fg, and 10 fg of DNA or no DNA using primers 16F and 16SR and Amplitaq LD that had been pretreated with Sau3AI. Lane 9 contained the 1-kb size marker (Promega). Lanes 10 to 17 contain the products of the amplification of 1 μl of each of the first-round samples with primers NF and NR. Lane 18 contains a reagent control for the second round of amplification.