CAJ | 학술논문

目的：改进一种以含蛋白酶K的裂解液作用于石蜡包埋处理的细胞、分离DNA粗提液用于PCR扩增的实验方案。方法石蜡包埋组织切片经常规脱蜡处理,用蛋白酶K裂解液洗脱细胞,55℃消化3 h后离心,吸取上清液；分光光度法检测DNA酶裂解液质量和浓度；以之为模板分别扩增人线粒体基因微卫星多态D310区、ATPase6基因区和核基因HBB、BRCA1等的DNA片段,琼脂糖凝胶电泳检测分析PCR扩增情况,并测序验证扩增产物。结果以石蜡切片的DNA裂解粗提液为模板,扩增目的DNA片段阳性率可达100%；获得序列分析验证。结论改进的蛋白酶K裂解液一步洗脱消化法操作简单、过程快捷、费用低廉,能快速有效地分离石蜡组织切片DNA,用于后续PCR扩增检测,具有较高效率。
목적：개진일충이함단백매K적렬해액작용우석사포매처리적세포、분리DNA조제액용우PCR확증적실험방안。방법석사포매조직절편경상규탈사처리,용단백매K렬해액세탈세포,55℃소화3 h후리심,흡취상청액；분광광도법검측DNA매렬해액질량화농도；이지위모판분별확증인선립체기인미위성다태D310구、ATPase6기인구화핵기인HBB、BRCA1등적DNA편단,경지당응효전영검측분석PCR확증정황,병측서험증확증산물。결과이석사절편적DNA렬해조제액위모판,확증목적DNA편단양성솔가체100%；획득서렬분석험증。결론개진적단백매K렬해액일보세탈소화법조작간단、과정쾌첩、비용저렴,능쾌속유효지분리석사조직절편DNA,용우후속PCR확증검측,구유교고효솔。
Objective To improve a simple method to extract DNA from tissues fixed in paraffin sections by direct digestion with proteinase K lysis buffer. Methods Formalin-fixed paraffin-embedded sections were routinely deparaf-finized and then the tissues were washed off for the glass slides using digestion buffer with proteinase K and transferred into a 1. 5 mL microtube for each sample. After incubation at 55 ℃ for 3 h,the supernatant containing DNA was transferred into a new tube. The DNA concentration and A260/280 values of the lysate were assayed by ultraviolet spectrophotometry. Amplifi-cation of the mitochondrial DNA fragment flanking D310 microsatellite region,part of the ATPase6 gene as well as nuclear genes HBB and BRCA1 were performed,and PCR amplification following detection with agarose gel electrophoresis was ana-lyzed. Results As shown by agarose gel electrophoresis and DNA sequence analysis, all the target mtDNA fragments were successfully amplified by PCR using the DNA lysate directly as template. Conclusion A simple-manipulation,rap-id-procedure and low-cost method for DNA separation from paraffin sections was presented here,by which the DNA content was extracted by one-step digestion with proteinase K lysis buffer;its effectiveness were evidenced by PCR of mitochondrial DNA and nuclear DNA fragments.