一步法分离石蜡切片DNA的改进与鉴定

DNA Isolation from Paraffin Sections by an Adjusted One-Step Protocol and its Qualification Test by PCR

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。

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.