摘要
目的建立从甲醛固定石蜡包埋组织中高效、快速提取基因组DNA的方法。方法将一片10μm厚石蜡包埋大肠癌组织切片直接浸入150μl消化液中,56℃孵育0.5h、1h和2h,灭活蛋白酶K后离心取上清即为DNA提取物。琼脂糖凝胶电泳检测所提取DNA的片段分布,紫外分光光度法测定DNA的产量及纯度。以提取的DNA为模板,PCR扩增不同长度产物片段并进行产物的直接测序以评价提取方法。结果该提取方法能够获得较长片段的基因组DNA,从一片10μm厚石蜡切片中即可获得大约60μg的DNA,在0.5h消化条件下获取的模板DNA,即可实现对152bp、293bp、392bp和541bp等4种不同片段的100%扩增,1h消化组的PCR产物可直接用于DNA测序。结论建立了一种简单、快速地从甲醛固定石蜡包埋组织中提取DNA的方法,只需一步消化即可获得满足普通PCR及测序需要的基因组DNA。
Purpose To establish a powerful one-step method for rapid DNA extraction from formalin-fixed paraffin-embedded tissues.Methods One 10 μm archival tissue section was directly placed into the digestion buffer without any pretreatment and incubated at 56 ℃ for 0.5 h,1 h and 2 h respectively,and then submitted to inactivation of proteinase K and centrifugation.Genomic DNA in the supernatant was quantified and qualified by DNA electrophoresis,spectrophotometry and used as the template of polymerase chain reaction,followed by DNA sequencing.Results This method extracted about 60 μg of DNA with high molecule weight from one 10 μm section,and produced a 100% amplification success rate for fragments of 152 bp,293 bp,392 bp and 541 bp even though in the 0.5 h group.Sequencing results of PCR products in 1 h group showed low level background noise and satisfied signal-to-noise ratio.Conclusions This simplified and cost DNA extraction method can extract high-yield DNA rapidly from formalin-fixed paraffin-embedded tissues and achieve specific PCR products available to meet the need of DNA sequencing.
出处
《临床与实验病理学杂志》
CAS
CSCD
北大核心
2010年第5期590-593,共4页
Chinese Journal of Clinical and Experimental Pathology
基金
国家自然科学基金(3090172)
辽宁省教育厅高等学校重点实验室项目(20060907)
辽宁省教育厅青年基金(05L558)
