HLA-DQalpha Typing by in situ PCR

Research Project

Project/Area Number	09670443
Research Category	Grant-in-Aid for Scientific Research (C)
Allocation Type	Single-year Grants
Section	一般
Research Field	Legal medicine
Research Institution	Oita Medical University
Principal Investigator	KISHIDA Tetsuko Oita Medical UniversityFaculty of Medicine Associate Professor, 医学部, 助教授 (50136793)
Co-Investigator(Kenkyū-buntansha)	FUKUDA Masako Oita Medical UniversityFaculty of MedicineResearch Associate, 医学部, 助手 (00156788) TAMAKI Yoshihiro Oita Medical UniversityFaculty of MedicineProfessor, 医学部, 教授 (20028377)
Project Period (FY)	1997 – 1998
Project Status	Completed (Fiscal Year 1998)
Budget Amount *help	¥3,200,000 (Direct Cost: ¥3,200,000) Fiscal Year 1998: ¥800,000 (Direct Cost: ¥800,000) Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Keywords	In situ PCR / Buccal cell / STR / DYS389II
Research Abstract	In situ PCR requires pre-treatment of samples in order to get penetration of the PCR components into the cells and allow the target sequences to be accessed for amplification. The permeabilization must be carefully controlled so that tissue morphology is maintained and the larger amplicons do not diffuse out. The most popular pre-treatment with proteinase K depends on the type of specimen and the fixation method. We explored for simple and reproducible pre-treatment without enzyme digestion. We used buccal cells as a model and amplified the DYS389II locus on the Y chromosome. After washing buccal swabs with distilled water and boiling them on a waterbath for 5 min., we amplified the locus with biotin-labeled primers in a microtube. We detected PCR products with alkaline phosphatase-conjugated streptavidin. Alternatively, we used formalin-fixed buccal cells. In male samples, we were able to detect typical positive cells whose nuclei were strongly stained. However, the cytoplasm of some cells was also stained. On the other hand, female samples showed no positive signal. Cells fixed with formalin gave similar results. These findings indicate that fixation/disruption by boiling is effective as pre-treatment for in situ PCR. We have spent most of the time for preliminary experiments. Although we were not able to achieve the original purpose, HLA-DQ alpha typing by an situ PCR, we successfully simplified pre-treatment of samples in a decisive manner. After the term of the project, we will make, an effort to develop HLA-DQ alpha typing by in situ PCR.