A simple and efficient PCR method was developedfor generating dye- or radiolabeled single-stranded DNA targets or probes used for hybridization studies. The method involved the use of a pair of long primers with high annealing temperatures and a short, labeled primer with a low annealing temperature in a PCR consisting of two cycles at different temperatures. We used this method to generate dye Cy™ 5-labeled and [³²P]-radiolabeled single-stranded DNA targets and probes. These labeled probes were used successfully for the microarray identification of point mutations in Mycobacterium tuberculosis genes and for the Northern blot detection of expression changes of the GATA-2 gene in Pneumocystis carinii-infected rat lungs.

Introduction

The creation of amplification methods to generate single-stranded DNA (1,2) has represented a major advance in development of PCR technology. Single-stranded DNA has been shown to be very useful for DNA hybridization studies (3) with a highly efficient hybridization and no need to be denatured before hybridization. Single-stranded target DNAs have been efficiently used in the studies of micro-array hybridization (4,5,6,7) and direct sequencing of DNA (1,8).

Single-stranded DNA can be generated by conventional asymmetric or real-time asymmetric PCR (9,10). Alternative methods of generating single-stranded DNA targets and probes include the utilization of biotin-streptavidin purification procedures (11,12). However, many of the methods of single-stranded DNA generation require extra steps to process the products and thus are time-consuming and costly. Conventional asymmetric PCR procedures using an unequal concentration of the forward and reverse amplification primers are theoretically simple (13), but they did not give us satisfactory results as other researchers have indicated (10).

The purpose of this study was to develop an improved asymmetric PCR procedure for generating single-stranded DNA. This novel method used a unique PCR nesting approach to generate single-stranded DNA probes. We demonstrated that these probes were effective for the DNA hybridizations associated with two different molecular biology procedures. The single-stranded DNA targets that we generated were used for the microarray detection of mutations in several drug-resistant strains of Mycobacterium tuberculosis. We also generated single-stranded DNA probes for use with a Northern blot procedure involving detection of the expression of a transcription factor, GATA-2, associated with Pneumoncystis carinii infection. Overall, the method that we developed for asymmetric PCR generation of labeled, single-stranded probes or targets is relatively easy to perform and was effectively utilized to create DNA probes for two different biological assays.

Materials and Methods

Oligonucleotide Primers and Probes

The design of PCR primers and probes used for microarray analysis was based on the published genome sequence of M. tuberculosis (14) and published data on the M. tuberculosis gene mutations associated with drug resistance (15). The DNA primers and amine-linked probes were synthesized by the Center for Biologics Evaluation and Research's Facility for Biotechnology Resources (U.S. Food and Drug Administration, Rockville, MD, USA). They were prepared with a Model 394 automated DNA synthesizer (Applied Biosystems, Foster City, CA, USA) using cyanoethyl phosphora-midite nucleosides. The probes we designed and used for microarray studies included wild-type AOK463: 5′-TCCCGATGCCCGGATCTG-3′; mutant AOK463: 5′-TCCCGATGCC AGGATCTG-3′; wild-type AOK587: 5′-GCTCCAGCACGGCAAAGG-3′; and mutant AOK587: 5′-GCTCCA TCACGGCAAAGG-3′.

Asymmetric PCR for Microarray Analyses

Double-stranded DNA templates containing point mutations in the M. tuberculosis gene katG were prepared by a recombinant PCR in vitro mutagenesis technique (16,17,18). Single-stranded DNA targets were then generated by our asymmetric PCR technique (Figure 1). This technique used three primers in a PCR. Two paired primers (forward and reverse), with a high annealing temperature (70°C) and a very low concentration (1 pmol/40 µL), were used for the generation of a small amount of primary double-stranded PCR product in the first round of PCR. A short Cy™ 5-labeled third primer, with a much lower annealing temperature (54°C) and a high concentration (15 pmol/40 µL), was designed to nest inside of the products generated in the first round of PCR to create single-stranded targets or probes in the second round of PCR. The forward and reverse primers, MTB-Kf2 (5′-CAAG CTGATCCACCGAGACATG-3′) and MTB-Kr2 (5′-CTTGTCGAG CAGCATGTACTCG-3′), were used to generate a 610-bp PCR fragment containing codon positions of 463 and 578 in the katG gene. The Cy5-labeled third primer, Cy5-Kf2s (5′-ATCCACCGAGACATGG-3′), nested inside of the PCR product generated by MTB-Kf2 and MTB-Kr2. For M. tuberculosis mutation detection, each 40 µL of PCR solution consisted of PCR buffer (2 mM Tris-HCl, pH 8.0, 1.5 mM MgCl₂, 10 mM KCl, 10 mM EDTA), 200 µM each dNTP, 1 pmol each forward and reverse primers, 15 pmol Cy5-labeled third primer, 50–60 pg recombinant PCR-generated template, and 2 U Takara Taq DNA polymerase (Takara Mirus Bio, Madison, WI, USA). The cycling program was 94°C for 3 min for DNA denaturation, followed by 20 cycles of 94°C for 20 s, and 70°C for 90 s. This was followed immediately by 20 cycles of 94°C for 20 s, 54°C for 20 s, and 72°C for 1 min. To test whether the PCR products consisted of single-stranded DNA, the PCR product was treated with S1 nuclease (20 µL PCR product/20 U S1 nuclease at 37°C for 30 min). Doubled-stranded PCR product generated with the first pair of primers was also treated at the same conditions as a comparison. Five micro-liters of each treated and untreated PCR product were electrophoresed side-by-side on a 1% regular agarose gel in TBE buffer (Roche, Indianapolis, IN, USA) at 150 V to compare the existence of single-stranded fragments. The gel was stained with ethidium bromide in TBE buffer for 30 min before photography under UV light. All the PCR-generated products to be used for hybridization (targets) were mixed together, purified by ethanol precipitation, and dissolved in 60 µL distilled water.