[Study of the PCR conditions on a slide for in situ PCR].
In order to find the conditions for in situ PCR, we examined the efficiency of PCR in a solution phase on the slide. DNA was extracted from the cells transfected with HCV-NS3 region. The primers were HCV-NS3 specific oligonucleotides. The results showed that the condition of DNA denaturation on the slide was suited at 90-92 degrees C for 10-60 seconds. In case of over 94 degrees C, DNA was not amplified and it suggests that the activity of Taq-polymerase was lost during the denaturation. As regards annealing temperature and times, if the melting temperature (Tm) was used for the annealing, we needed relatively longer times, and if annealing temperature was about 5 degrees C lower than the Tm, it was enough to use relatively short times. In addition, a favorable result was obtained when in situ PCR was carried out under the best conditions for PCR on a slide. It is indicated that the conditions for general PCR in a tube is not applicable to the amplifying of cellular DNA targets on a slide for detection in situ.
Yamashita
M
M
Department of Pathology, Kanazawa Medical University, Ishikawa.
Takegami
T
T
Sasaki
K
K
Nojima
T
T
D000818:Animals; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D016174:Hepacivirus; D051379:Mice; D016133:Polymerase Chain Reaction / Q000379:methods*; D012996:Solutions; D013696:Temperature; D013997:Time Factors; D014162:Transfection
D012996:Solutions; D004247:DNA