Application of Hot Start PCR Method in PCR-based Preimplantation Genetic Diagnosis

PURPOSE: To determine a method to improve the efficacy and accuracy of preimplantation genetic diagnosis (PGD) - polymerase chain reaction (PCR), we compared hot start PCR and conventional multiplex nested PCR. MATERIALS AND METHODS: This study was performed with single lymphocyte isolated from whole blood samples that were obtained from two couples with osteogenesis imperfecta (OI). We proceeded with conventional multiplex nested PCR and hot start PCR in which essential reaction components were physically removed, and we compared the amplification rate, allele dropout rate and nonspecific products. Afterward, we used selective method for PGD. RESULTS: In the two couples, the respective amplification rate were 93.5% and 80.0% using conventional multiplex nested PCR and 95.5% and 92.0% using hot start PCR. The respective mean allele dropout rates for the two couples were 42.0% and 14.0% with conventional multiplex nested PCR and 36.0% and 6.0% with hot start PCR. CONCLUSION: The results demonstrate that the hot start PCR procedure provides higher amplification rates and lower allele dropout rate than the conventional method and that it decreased the nonspecific band in multiplex nested PCR. The hot start method is more efficient for analyzing a single blastomere in clinical PGD.

Detection of Herpes Simplex Virus Type-1 DNA by In Situ Polymerase Chain Reaction

BACKGROUND: Standard solution-phase PCR cannot localize the amplified DNA products in cells or tissue sections. Recently, in situ PCR technique which combines PCR with in situ hybridization was developed and applied to detect target DNA or gene expression in the tissue sections. OBJECTIVE: The purpose of this study was to detect the presence of HSV type-1 DNA in herpes simplex lesions by using hot start PCR in situ hybridization and hot start in situ PCR and to compare the sensitivity and specificity of the two methods. The sensitivity and specificity of multiple overlapping primers and a single primer pair in hot start in situ PCR were also compared. METHODS: We performed hot start PCR in situ hybridization and in situ PCR with multiple overlapping primers, and hot start in situ PCR with a single primer pair in paraffin-embedded, formalin-fixed tissues. RESULTS: HSV type-1 DNA was detected in 4 (80%) of.5 cases of herpes simplex and negative in all cases of herpes zoster, verruca vulgaris, and normal skins. One negative case of herpes simplex could not be detected by HSV type-1 specific primers because it might be caused by HSV type-2. There was no difference in the sensitivity, specificity, and intensity of signals between the three methods. CONCLUSION: Hot start in situ PCR with a single primer pair is a simpler, easier, and more rapid technique for detecting the HSV type-1 DNA in lesional tissue sections with similar sensitivity and specificity than hot start PCR in situ hybridization and hot start in situ PCR using multiple overlapping primers.