福尔马林保存的动物标本基因组DNA的提取方法

A METHOD OF EXTRACTING GENOMIC DNA FROM ANIMAL SPECIMEN PRESERVED IN FORMALIN

从在福尔马林长期保存的动物物标本中提取基因组DNA用于分子生物学研究一直是一个未得到彻底解决的难题。本文提出了一种从浸泡在福尔马林的大仓鼠肝脏和日本鳗鲡肌肉标本中提取基因组DNA的新方法。取浸泡于福尔马林中的大仓鼠肝脏或日本鳗鲡肌肉适量 ,用PBS溶液冲洗 ,放在灭菌的吸水纸上将其揩干 ,于超净工作台内用无菌剪刀将材料剪成 5 0mg的小块 ,放入PBS液浸泡 12～ 2 4h ;然后转入 70 %的乙醇中处理 12～ 2 4h。依次换入下列梯度酒精中处理 :80 %乙醇 ,2h ,重复一次 ;90 %乙醇 ,2h ,重复一次 ;95 %乙醇 ,2h ,重复一次 ;10 0 %乙醇 ,1h ,重复一次。然后将材料放入 1/ 2倍的PBS液中浸泡 12h ,其间更换一次溶液。提取方法参考Sambroock等人 (1989) ,加蛋白酶K的量按标准量 (10 0 μg/mL) ,在 5 0～ 5 6℃温浴 3～ 6h处理过程中 ,不断轻摇混匀 ,视消化效果可以重复加入标准量的 1/ 2倍蛋白酶K进行消化 ,直至将材料完全消化为止 ;酚氯仿抽提最后一次的上清液移入透析袋中透析 ;沉淀DNA时 ,在 - 2 0℃下 2 0min效果为宜。该方法主要特点在于对标本进行预处理 ,在保证不使DNA进一步降解的前提下 ,首先去除标本中所含的福尔马林溶液的成分 ,然后利用改进的酚氯仿抽提法提取该类标本的基因组DNA 。

The extraction of genomic DNA from long preserved animal specimen fixed with formalin and the application of this kind of DNA in molecular research is an unsettled problem until now. This paper reports a new method to extract genomic DNA from the liver specimen of rat like hamster (Cricetulus triton) and muscle specimen of Japanese eel (Anguilla japonica) preserved in formalin. First, took some liver tissue of the rat like hamster (Cricetulus triton) and muscle tissue of the eel (Anguilla japonica); second, cleaned them with PBS solution; and dried them with sterilized water absorbing paper; third, cut them into the pieces about 50mg weight; and put the pieces into PBS solution for 12～24 hours; fourth, flooded them in two changes of 70% alcohol for 12～24 hours each, then infuse them into alcohol as the following concentration: in two changes of 80% alcohol for 2 hours each, two changes of 90% alcohol for 2 hours each, two changes of 100% alcohol for 1 hour each, after all of the above treatment, put the samples into 1/2 PBS solution for 12 hours, during this treatment, changed the 1/2 PBS solution once. The extraction method was based on that described by Sambrook et al. (1989) with modification. The final concentration of proteinase k was 100 μg/ml. During the 3～6 hr\|incubation in water bath, the temperature was kept at 50℃ to 56℃, and the reaction tube was taken out and shook slightly again and again. If the specimen tissue was not digested well, more proteinase k must be added to the solution (usually half of the standard quantity) until all the tissue was digested completely. After the last extraction by phenol and chloroform, the supernatant fluid was transferred into dialysis bag. When deposited, the DNA should be deposited at -20℃ for 20 minutes. The key to this method was the pretreatment of the specimen. First, remove the formalin from the specimen under the condition that the genomic DNA is not further degraded. Second, extract the genomic DNA from the specimen with the improved phenol and chloroform extraction method. Finally, purify the genomic DNA with dialysis bag. The result showed that the purified DNA could be used in PCR using random primer, the amplification of microsatellite site, southern and dot blotting.