Long-term storage of unamplified complete PCR mixtures.

The polymerase chain reaction (PCR) (2) has evolved into one of the most powerful and widely used tools in molecular biology. Apart from its application in basic research, the universal nature of the PCR technique has proven extremely useful in the field of medical microbiology for the diagnosis and surveillance of infectious diseases. Although the technique is distinguished by its outstanding sensitivity and specificity, in some fields of application the consistency of results obtained on clinical samples is hampered by intrinsic factors like pipetting deviations or variable amplification conditions. To rule out variations in the concentration and the properties of the components, it would be advantageous to assemble individual PCR mixtures in advance and store them, ready for use, for prolonged periods. In routine practice, one encounters several diagnostic situations where comparability of the results is of utmost importance but whereby only a limited number of amplifications has to be carried out at a given time. When performing arbitrarily primed (AP)-PCR (9, 10), for example, the availability of PCR mixtures with an identical composition turns out to be the key factor in obtaining reproducible fingerprints. Moreover, with respect to the extensive PCR repertoire of diagnostic institutions, it is time-consuming, laborious and often impracticable to prepare individual PCR mixtures at each occasion. We report the performance of preassembled PCR mixtures that had been stored at either -20° or -70°C for different periods, both in amplifying a 1497bp fragment of the 16S rRNA gene of Acinetobacter baumannii (Catalog No. 19606T; ATCC, Rockville, MD, USA) and in generating DNA fingerprints by the use of AP-PCR on a genetically identical Legionella pneumophila clinical isolate. Due to the time-consuming sample processing procedures associated with pulsed-field gel electrophoresis (PFGE), the application of AP-PCR gained more and more importance in the field of molecular epidemiology. Next to its simple execution and the undemanding nature regarding the sample DNA, it provides the potential to analyze most of the medically important organisms by a single technique (4). In the course of AP-PCR, genomic DNA is amplified under low-stringency conditions. Using a single, short oligonucleotide primer of arbitrary sequence, DNA synthesis is allowed to initiate from sites to which the primer is fortuitously and only partially matched. The fragments generated are probably drawn from throughout the genome, each reflecting the presence of two fortuitous primer-binding sites within reach of each other (usually less than 3 kb apart). Thus it is likely that polymorphisms in the fragment patterns result from insertions and deletions between the primer-binding sites or other mutations at the primer-binding sites. Because the fragment generation is a fortuitous process, the reproducibility of AP-PCR patterns evidently varies depending on the procedure of DNA extraction, the selected primer sequences and the amplification parameters. Using HPLC-purified primer oligonucleotides in combination with standardized PCR mixtures, one can rule out the main causes of deviation during the amplification procedure. Still, run-to-run variability and limited inter-laboratory reproducibility remain well-known problems of AP-PCR (3,5,6). Although several commercial kits have been launched recently in aid of AP-PCR reproducibility (e.g., ReadyTo-Go RAPD Beads; Pharmacia Biotech, Freiburg, Germany), we investigated the performance of batch-prepared PCR mixtures that were stored frozen in aliquots until use. A mixture was prepared containing 6 mM primer AP (GGTGGTGGCT) (1), 200 U of AmpliTaq DNA Polymerase (PerkinElmer, Weiterstadt, Germany), 200 mM of each dNTP, 0.5 mL of 10× PCR buffer (100 mM Tris-HCl, pH 8.3, 15 mM MgCl2, 500 mM KCl and 0.01% gelatin) and ultrapure H2O (Sigma Chemical, Munich, Germany) up to a final volume of 5 mL. An equal mixture was prepared containing 10% (vol/vol) of glycerol as an additional component. Mixtures were stored in ready-to-use 50-μL aliquots at both -20° and -70°C. Performance of PCR mixtures was repeatedly tested after 1, 2, 3, 4, 8, 12 and 20 weeks of storage. AP-PCR analysis was carried out by adding 10 ng of DNA prepared from an L. pneumophila serogroup 1 isolate to a freshly thawed PCR mixture. The amplification reactions were performed in a GeneAmp PCR System 9600 Thermal Cycler (Perkin-Elmer) without oil