포르말린으로 고정된 간조직으로부터 C4 유전자의 Taq1 RFLPs

Abstract

Frequently we confront necessity of repeated and retrograde studies for DNA Frequently we confront necessity of repeated and retrograde studies for DNA information and family analysis from dead persons. So we report a method for the extraction of genomic DNA and its C4 RFLPS( restriction fragment length polymorphisms) from formalin fixed hepatic tissue. Associations of C4 allotypes and haplotypes with a number of disease have been reported, in addition, linkage disequilibrium with other MHC markers, the relationship between C4, Rg and Ch, as well as the hemolytic function of different C4 allotype patterns were investigated. 5 grams of the formalin fixed hepatic tissue were laird in tap water for 1 week, washed with 0.85% NaCl, and suspended in PBS. After'the suspension was incubated with agitation at 60oC for 2-3hrs, the tissue fragments were suspended in 500mM Tris. HCI(pH9) j 20mM EDTA/10mM NaCl. The suspension in which laA SDS and Img/ml proteinase K were added which was incubated with agitation at 50t for 24hrs, and was repeated for 50hrs, with same procedure. Genomic DNA was extracted with phenol l chloroform J ethanol method. After measuring the concentration of the DNA, its digestion was done by Taql enzyme. The DNA underwent Southern blot hybridization by using PAT-A(locus 6p21.3) as a probe. The results showed 3 kinds RFLPS (5.8/6.6/7.4)kb, (6.6/7.4)kb, (6.6/7.4)kb, By Okimoto reports(not published ) the longer storing period, the smaller sue of DNA was abt-ained, but the genomic UNAs extracted from 6-12 month-stored tissue showed clear Southern blots. The present mothod is applicable for the fancily analysis of genetic ninases and the detection of an oncogene.

하기의 방법으로 하여 formalin에 상당기간 고정된(6-12개월)간 조직에서 DNA추출 과정에 다소 긴 시간이 소요되었으나 높은 순도의 DNA를 얻었다. (260nm1280nm=1.84∼1.90) Taq 1 효소로 DNA를 자르고 PAT-A(C.) 유전자로 Southern blot hybridzation하여 3가지 유형의 polymorphic band를 얻었다. 즉(5.8/6.6/7.4)kb, (6.6/7.4)kb, (5.8/7.4)kb이다. 이로서 이미 사망한 가족으로부터 C.유전자의 DNA 정보를 얻을 수 있어 가족성으로 존재하는 유전성 질환이나 악성 종양의 진단에 좋은 자료가 될 것으로 기대된다.