角类动物药DNA提取方法研究

Study on extraction of DNA from animal cornu

目的建立角类动物药材的DNA提取方法,使提取的DNA质量满足PCR及测序要求,同时可用于陈旧动物角类样品的DNA提取。方法为避免取骨塞部位用EDTA脱钙造成DNA提取不完全或DNA被破坏,取角质层部位,采用二硫苏糖醇(DTT)结合化学试剂盒中的细胞裂解液及蛋白酶K处理角质层样本,其余按试剂盒操作提取DNA。考察了取样量、DTT用量对角质部位DNA质量的影响。结果确定了取样量为25 mg,DTT用量为20μL,角质细胞裂解完全,所有样品的DNA质量均可满足PCR要求。结论建立的DNA提取方法,提取DNA完全,可标准化操作,可应用于多种动物角的提取。

Objective To establish the method of DNA extraction for animal cornu, ensuring the quality of extracted DNA to meet the requirement of PCR and sequencing, which can be used in DNA extraction for the old animal horn samples. Methods To avoid incomplete DNA extraction or damaged DNA by using EDTA decalcify when drawing material from bone, DTT, cell lysis solution and proteinase K were used to treat horn cells when drawing material from horny layer, other steps of DNA extraction were operated according to the reagent kit instructions. The effects of sampling amount and DTT dosage on the quality of DNA from horny layer were also investigated. Results The sampling amount was 25 mg, DTT dosage was 20 μL, horn cells can be lysised completely, and the quality of DNA of all samples can meet the requirements of PCR. Conclusion The method of DNA extraction from animal cornu established in this study can extract DNA completely, ensure standardized operation, and could be applied to the extraction of various animal horns.