PCR Principles and Reaction Components

CHARACTERIZATION OF A DUPLEX POLYMERASE CHAIN REACTION ASSAY FOR THE DETECTION OF ENTEROTOXIGENIC STRAINS OF STAPHYLOCOCCUS AUREUS

“…In our study, a critical innovative step of the duplex PCR assay is the optimization of amplification conditions by uniform design, which ensures that all target fragments can be satisfactorily amplified with minimum trials. According to Rolfs et al . (1992), the key factors affecting PCR reactions can be summarized as Mg 2+ concentration and annealing temperature.…”

Molecular analysis of chronic granulomatous disease caused by defects in gp91-phox

“…A total of 500 ng of each sample was amplified after denaturation in 50 µl of reaction buffer containing 1× polymerase chain eaction (PCR) buffer (Boehringer Mannheim, Indianapolis, IN), 200 µM dNTP mix (Pharmacia, Piscataway, NJ), 2.6 ng/µl of each primer, and 2.5 U Taq polymerase (Boehringer Mannheim), using a GeneAmp 9600 thermal cycler (Perkin Elmer, Norwalk, CT). The conditions for the amplification reaction were 30 cycles of 96°C/ 5 sec, 60°C/15 sec, and 72°C/30 sec, followed by a 7-min extension at 72°C (Rolfs et al, 1992). Synthetic oligonucleotides for SSCP and genomic DNA sequencing (Table 1) were constructed using the known intronic sequences on either side of gp91-phox exons (Skalnik et al, 1991).…”

Reply

“…Biopsy specimens were stored at Ϫ70°C, and DNA was extracted and purified using QIAmp DNA Minikit reagents according to the instructions of the manufacturer (Qiagen, Crawley, UK). Extracted DNA was quantified by Hoechst H33258 dye binding (8), and its suitability for PCR analysis confirmed by amplification of the human single-copy gene, pyruvate dehydrogenase (PDH), as previously described (9). One microgram of the extracted DNA was tested by nested PCR for the presence of HRV-5 sequences, using the pol primer set and thermal cycling conditions described by Griffiths et al (2).…”