Development of tissue fixatives to minimize DNA fragmentation - DNA analysis for formalin-fixed and paraffin-embedded tissues -
Grant-in-Aid for Scientific Research (C).
|Research Institution||DOKKYO UNIVERSITY SCHOOL OF MEDICINE|
TAKAHASHI Masanori Dokkyo Univ.School of Med., Legal Medicine, Associate Professor, 医学部, 助教授 (70103356)
加藤 幸映 獨協医科大学, 医学部, 助手 (60245090)
|Project Fiscal Year
1997 – 1999
Completed(Fiscal Year 1999)
|Budget Amount *help
¥2,800,000 (Direct Cost : ¥2,800,000)
Fiscal Year 1999 : ¥500,000 (Direct Cost : ¥500,000)
Fiscal Year 1998 : ¥900,000 (Direct Cost : ¥900,000)
Fiscal Year 1997 : ¥1,400,000 (Direct Cost : ¥1,400,000)
|Keywords||formalin fixation / paraffin embedding / DNA / PCR / Gene analysis / EDTA / VNTR / STR / ホルマリン固定 / パラフィン包埋 / 遺伝子解析 / パラフィン切片 / ABO遺伝子 / アメロゲニン / パラフィン / AB0遺伝子型|
To minimize degradation of genomic DNA molecules with formalin as the tissue fixative, the additives of EDTA, sodium chloride, methanol and flavonoid were examined.
Human tissue specimen was soaked with 10%v/v phosphate-buffered formalin (pH 7.2) and various concentrations of these additives for 24 hours to two years.
Thin-sectioned specimen in five μm thick was deparaffinized with xylol and the DNA of five thin-sections in every sample was extracted by means of the phenol-chloroform method.
DNA samples extracted were electrophoresed in the agarose gel. Gels were trimmed at 1.2kbp in size.
Contents of DNAs extracted from respective gel strips were measured by UV absorbance. Then, these DNAs were amplified by PCR for the loci of D1S80 and some STRs.
The results were as follows :
For 7 days after fixation, DNAs of more than 1.2kbp decreased drastically to 20% with formalin alone, and to 42% with buffered formalin. EDTA and flavonoid improved 2-4% additively.
After the day, no large DNA was remained with formalin alone fixative.
Two years later, EDTA addition resulted to be 12% of 1.2kbp DNA content, while other additives were less than 7%. Methanol brought to be worse.
Even after 2 years, EDTA fixative gave good template of DNAs of the lung, the kidney and the liver for PCR-based genotyping of D1S80. In addition, TPOX, vWA and THO1 were possibly amplified by more than 500bp size template.
EDTA inhibited significantly DNase I activity in the tissues, which concentration was enough at 5mM.
Taken all together, EDTA and buffered formalin for tissue fixatives is preferentially recommended.
Research Output (7results)