A Hot Start Polymerase Chain Reaction (PCR) entails the withholding of at least one reagent from the reaction mixture until the reaction tube temperature has reached 60-80 degrees C. Hot Start amplification with an AmpliWax vapor barrier uses a layer of solid wax to separate the retained reagent(s) and the test sample from the bulk of the reagents until the first heating step of automated thermal cycling melts the wax and convectively mixes the two aqueous layers. Wax-mediated Hot Start PCR greatly increases the specificity, yield, and precision of amplifying low copy numbers of three HIV targets. In the presence of 1 microgram of human placental DNA (1.6 x 10(5) diploid genomes) the specificity improvement entails considerable to complete reduction in the amplification of mis-primed sequences and putative primer oligomers. When mis-priming is negligible, the procedural improvement still suppresses putative primer oligomerization. Hot Start PCR with an AmpliWax vapor barrier permits routine amplification of a single target molecule with detection by ethidium stained gel electrophoresis; nonisotopically visualized probing suffices for confirmation. The improved amplification performance is evident for target copy numbers below approximately 10(3).

Many artifacts during Taq DNA polymerase PCR have been reported, especially with PCR targets containing stable secondary structure (1, 2, 3, 4). The consequence with PCR targets containing stable secondary structure usually is either very low efficiency in PCR yield, or deletion mutagenesis in the PCR products (1, 2). Pallansch et al. have estimated that AG for such stable secondary structures calculated by the FOLD computer program is <-14 kcal/mol for RNA target (2). This kind of deletion mutagenesis is DNA polymerase dependent (1), and probably determined by polymerase processivity. The Taq DNA polymerase used in PCR has relatively low processivity (6). Highly processive enzymes may be able to overcome this problem. Interestingly, Cariello et al. have successfully used a thermolabile highly processive modified T7 DNA polymerase (SequenaseC) (5) to amplify a segment of 18S rRNA-encoding gene without PCR deletion mutagenesis (1). Here I described the use of E. coli single-strand DNA binding protein (EcoSSB) to minimize deletion mutagenesis artifacts during Taq DNA polymerase PCR. Same primers, DNA template and similar PCR conditions as Cariello et al. were used here to amplify a segment of 18S rRNA-encoding gene. The AG for this DNA segment calculated by the FOLD computer program is-33 kcal/mol (Figure 1A). As shown in Figure iB, the full length upper (389 bp) band was increased dramatically in presence of EcoSSB. The lower (335 bp) deleted PCR product was not changed in presence of 0.75 Ag EcoSSB, and was almost eliminated in presence of 1.5 jig EcoSSB. The lower band was confirmed to be the deleted PCR products of the upper band by Southern blot. SSB concentration played an important role in optimizing PCR. High concentrations of SSB can inhibit the PCR reaction while low concentrations of SSB have no effects on PCR. I have found the optimal molar ratio of EcoSSB versus total ssDNA concentration in PCR mixture (with respect to EcoSSB binding site size, 100 nt per SSB tetramer) to be near 0.7. Hypotheses for this effect are that either EcoSSB improves Taq DNA polymerase processivity, or EcoSSB melts PCR target secondary structure during PCR, allowing Taq polymerase read through.

Recently we reported that, in HL-60 cells, transcription of the formyl peptide receptor (FPR) gene can be up- and downregulated by agents that induce differentiation of HL-60 cells into neutrophils. To begin studying the mechanisms involved in regulation of FPR gene expression, we cloned two human cDNAs and the gene coding for FPR. The genomic clone (pINF14) contained a 14.5-kb insert. A 2.7-kb EcoRI fragment was obtained from pINF14 that hybridized with an FPR open reading frame probe. The EcoRI fragment was sequenced and found to contain an intronless FPR open reading frame. Sequence alignment of the EcoRI genomic fragment with the FPR cDNA revealed that the first 31 bases of 5' untranslated FPR cDNA were not represented in the genomic fragment. Furthermore, a splicing consensus sequence was present in the genomic fragment at the site of divergence with the cDNA sequence. Restriction mapping and Southern blot analysis identified a 121-bp fragment that contained the sequence corresponding to the first 31 bases of 5' untranslated FPR cDNA. An additional (previously undescribed) 15-bp cDNA sequence in the 5' end of FPR were identified using an anchored polymerase chain reaction. This sequence was also contained in the genomic 121-bp fragment. This 121-bp fragment was located 5.2 kb (intron) upstream of the FPR open reading frame. It contained an unusual TATA box and displayed transcriptional activity in vitro and in vivo. Potential binding sites for AP-1 and glucocorticoid receptor were identified upstream of the putative TATA box.(ABSTRACT TRUNCATED AT 250 WORDS)

Effective sanitation of equipment is critical in an integrated food safety system. Existing sanitation verification methods have significant shortcomings that decrease their effectiveness and create an opportunity for novel approaches. This study is intended to validate abiotic bacterial surrogates (saniTracers™) for the rapid verification of solid surface sanitation processes. AOAC Validation studies included a pure analyte LOD, matrix studies, inhibition, selectivity, product consistency and stability, instrument variation and robustness. 4” x 4” stainless steel coupons, Pure or diluted analyte and Chai qPCR system were used for those studies. The saniTracers were quantified by qPCR. Average ΔCt values for all matrixes at 50ppm and 250ppm sodium hypochlorite sanitation show 3.23 and 11.95, respectively. saniTracers behave similarly in the presence of all matrixes. saniTracers were not inhibited at significant concentrations by any of the sanitizers used. No discrepant results were observed with the robustness study. Lot-to-lot/stability testing demonstrated no differences between the three lots of tests analyzed. saniTracers demonstrated high repeatability and minimal interference from matrix, disinfectant or microorganisms. The microbial selectivity study indicated a correlation between the removal of pathogens and change in Ct value for saniTracers after following the disinfection process. An average log(cfu) reduction of 4.00 was observed for the three pathogens evaluated correlating to an average 10.65 Ct change in saniTracers, indicating a correlation in the removal of an average of 0.38 log(cfu) of pathogens per one saniTracers Ct change after following the disinfection process.