PCR products of 1.8 kb were generated with DNAs from all Escherichia coli H7 strains tested by using oligonucleotide primers which flank the fliC gene. Three RsaI digestion profiles of these PCR products were evident on agarose gels; the first occurred with serotype O55:H7, O157:H7, or nonmotile (NM) strains, the second occurred with serotype O1:H7 and O18:H7 strains, and the third occurred wit...

BACKGROUND Splicing by overlap extension (SOE) PCR is used to create mutation in the coding sequence of an enzyme in order to study the role of specific residues in protein's structure and function. OBJECTIVES We introduced a nested-SOE-PCR (N -SOE-PCR) in order to increase the specificity and generating mutations in a gene by SOE-PCR. MATERIALS AND METHODS Genomic DNA from Bacillus thermoc...

A method is described for quantifying polymerase chain reaction (PCR) products by labeling them with biotinylated dUTP or dATP during the amplification step. The biotin-PCR products are size-separated by gel electrophoresis, transferred to a nylon membrane and then complexed with streptavidinalkaline phosphatase. The complexes are detected by reaction with a chemiluminescent substrate sheet. Th...

RT-PCR amplification of P450 2C6 from rat liver, using primers in opposite orientations of exon 6, resulted in PCR products containing segments of exons joined at non-consensus splice sites. Moreover, many of the PCR products identified were composed of not only a single region containing exonic segments joined at non-consensus splice sites but, instead, of several repeats of the non-canonicall...

A mathematical model and a computer simulation were used to study PCR specificity. The model describes the occurrences of non-targeted PCR products formed through random primer-template interactions. The PCR simulation scans DNA sequence databases with primers pairs. According to the model prediction, PCR with complex templates should rarely yield non-targeted products under typical reaction co...

bromide staining in detecting PCR products (Figure 3). Moreover, as expected, band intensities increase with longer duration of exposure to X-ray film (data not shown). Biotin-21-dUTP and biotin-14-dATP work equally well in labeling the PCR products (data not shown). Similarly, DNA cross-linking with baking and UV irradiation work equally well in this system (data not shown). We have also confi...

This method for rapid, automated analysis of polymerase chain reaction (PCR) products makes use of PCR primers containing 5'-polypyrimidine sequences. Polypyrimidine-"headed" primers confer to the PCR product the ability to form triple helical complexes with a third polypyrimidine oligonucleotide. Third-strand oligonucleotides are modified to serve as either capture reagents or detection reagen...