Development of high-performance in situ PCR method for gene detection and expression analysis

Development of in situ PCR method for detection of DNA/mRNA was made based on in situ hybridization on chromosome, cell and tissue after PCR amplification. In 1997, the final year of this project, amplification of the domain that codes btDYZ, a male-specific base sequence on Y-chromosome was examined by in situ PCR using 32P-labelled dCTP on the chromosome. The chromosome was prepared by 72 hours incubation of the blood taken from a male calf and conditions for preparation of a stable chromosome specimen were investigated. It was demonstrated that both fixation and development methods were able to produce satisfactory specimens, but air-drying method did not. As the number of PCR cycles increased, the stronger signals were produced, but there also appeared signals due to some non-specific products. Frozen specimens of bovine ovary were prepared and the conditions to obtain reliable results by in situ RT-PCR method were investigated using a bovine actin primer (bActin). The present results were almost same as those obtained with DIG-11-dCTP in 1996. The optimum temperatures for annealing and denaturation of DNA were 64degC and 94-98degC, respectively, higher by each several degrees than the respective conventional temperatures for RT-PCR. It was demonstrated that a combined method of RT-PCR and in situ hybridization was available for in situ detection and expression of a gene on chromosome. (M.N.)