Introduction

When analyzing RNA by reverse transcription PCR (RT-PCR) procedures, the elimination of contaminating DNA is of fundamental importance. Otherwise, the products obtained in PCR might originate from genomic DNA rather than specifically from cDNA produced from messenger RNA (mRNA). Removal of contaminating DNA is commonly achieved by treating samples with RNase-free DNase. However, DNase treatment is not well suited to a number of applications. The purpose of this study was to develop a new method to eliminate DNA-mediated product formation in RT-PCR by means of a probe specifically blocking amplification of contaminating DNA without affecting amplification of cDNA. Unlike eukaryotic mRNA, a poly(A) tail is not added to the 3′ end of bacterial mRNAs. This makes the design of RT-PCR primers that target mRNA without targeting the corresponding DNA sequence impossible. However, unlike the corresponding chromosomal DNA sequence, the cDNA has two defined ends, one corresponding to either the RNA 5′ end at the transcription initiation site or an RNA 5′ end generated by RNA processing and a 3′ end generated by the primer used in the reverse transcription procedure. By contrast, the corresponding chromosomal DNA sequences continue beyond these points and have ambiguous ends based on the fragment length of each molecule. Herein, we demonstrate that sequence-specific blocking probes (1) can be designed, targeting the region immediately upstream of and proceeding through, these cDNA endsequences. Selective inhibition of PCR amplification of the chromosomal DNA is achieved by introducing a preprimer annealing step at a temperature sufficiently high to allow a peptide nucleic acid (PNA) blocking probe to anneal to the genomic DNA but not to cDNA. The PNA probe will hereafter block attachment of a cDNA-targeted primer to the genomic DNA sequence, when the temperature is lowered to the primer annealing temperature. Under these conditions, the blocking probe will not affect attachment of the PCR primers to the cDNA. The blocking probe thereby prevents PCR amplification of genomic DNA without affecting amplification of RNA-derived cDNA (Figure 1).

Figure 1. Peptide nucleic acid (PNA) inhibitor probe principle. (Click to enlarge)

For this approach to work, probes should meet the following requirements: (i) the probe should not itself function as primer; (ii) the probe should exhibit a higher melting temperature (T_m) value than the corresponding PCR primer; and (iii) the probe should, when attached to the target sequence, efficiently prevent primer attachment. PNA bind tightly to complementary sequences of DNA, and the thermal stability of PNA/DNA duplexes at physiological ionic strength is generally higher per base pair than the corresponding DNA/DNA duplex (2,3). Further, PNA does not function as a primer for DNA polymerase and was therefore used in the present study. Probes consisting of PNA have previously been used successfully to eliminate PCR product formation in a sequence-specific manner (1,3,4,5). However, all probes previously used in attempts to block specific PCRs have been placed within the PCR-amplified sequence, inhibiting a fragment of differing sequence from the PCR target. This strategy is not suited for the present purpose.

To our knowledge, this study represents the first attempt to block a specific PCR by placing a probe partly outside the sequence of interest. We show that the PNA probe inhibition assay is reproducible and that amplification of cDNA templates is not inhibited.

Materials and Methods

Strains and Culture Conditions

Three bacterial strains, Escherichia coli SP308/pKJK10 (6), Pseudomonas putida OUS82 UCB55 (7), and Salmonella typhimurium LT2 (8) were used in this study. They were grown in appropriate media facilitating expression of the relevant genes.

DNA Extractions

DNA was extracted from pure cultures using commercial kits [Fast Soil DNA purification kit (BIO101, Vista, CA, USA) or UltraClean™ Soil DNA Extraction kit (MO-BIO, Solana Beach, CA, USA)] according to the manufacturer's instructions except that soil samples were replaced by pure culture samples.

RNA Extractions

Disposables and reagents were either certified RNase-free, or diethyl pyrocarbonate (DEPC)-treated (0.1%; Sigma, St. Louis, MO, USA) was added prior to autoclaving. RNA was extracted from approximately 10⁷ cells (200 µL, A₆₀₀ = 0.1) P. putida OUS82 UCB55 overnight culture; from 300 µL S. typhimurium overnight culture; and from 300 µL E. coli SP308 cells either grown to stationary phase. Total RNA was extracted using the FastRNA® Pro Blue kit (BIO101) in accordance with the manufacturer's instructions.