指甲游离缘及毛发中核DNA抽提方法的探讨

Evaluation of methods for human nuclear DNA extraction from free margins of finger nail and hair samples

目的探讨指甲游离缘及毛发中抽提核DNA的方法,并对抽提结果进行评估。方法采集5名健康成人志愿者指甲游离缘、发根及发干样本各30份。分别用无水乙醇和蒸馏水浸泡样本,去除外源性DNA。每份指甲游离缘样本为3mg指甲游离缘碎片;每份发根或发干样本各为3段0.3～0.5cm发根或发干。分别采用酚氯仿法、Chelex-100法和QIAamp DNA Investigator试剂盒法抽提3种样本的核DNA,并运用PCR扩增对抽提DNA进行评估。扩增片段位于不同染色体上,长度分别为188、248、300和741bp。PCR扩增产物用1.8%琼脂糖凝胶电泳检测。为探讨样本量对抽提结果的影响,任意取指甲游离缘1、3和5mg,发根及发干各1、3和5根重复实验,并比较结果。初步探讨增加Taq酶量对黑色素抑制的消除作用。结果在3种抽提方法中长度为248bp的引物扩增成功率均为最高。指甲游离缘样本使用试剂盒法抽提核DNAPCR扩增成功率显著高于酚氯仿法及Chelex-100法(P<0.05)。发干样本使用试剂盒法抽提核DNAPCR扩增成功率显著高于酚氯仿法(P<0.05)。发根样本3种方法抽提核DNAPCR扩增成功率差异无统计学意义(P>0.05)。指甲游离缘样本的PCR扩增成功率显著高于发根及发干样本(P<0.05),发根和发干样本的PCR扩增成功率差异无统计学意义。试剂盒法抽提指甲游离缘、发根和发干核DNAPCR扩增成功率随样本量增加而提高。酚氯仿法和Chelex-100法抽提发根或发干样本核DNAPCR扩增成功率随样本量增加呈下降趋势,提示可能存在黑色素抑制作用。增加Taq酶量对于消除黑色素抑制有一定作用。结论①3种抽提方法均能成功从指甲游离缘及毛发中抽提到核DNA,试剂盒法成功率最高。②指甲游离缘、发根和发干均可作为核DNA采样源,从指甲游离缘抽提DNA成功率最高。

Objective To evaluate three different assays,including the phenol-chloroform method, the Chelex-lO0 method, and the QIAamp@ DNA Investigator Kit method（ kit method） for extracting nuclear DNA （nDNA） from free margins of finger nail and hair samples. Methods 30 samples of free nail clipping margins, hair roots and hair stems were obtained from 5 healthy Chinese volunteers. All samples were washed by 100% ethanol and sterile water to remove extraneous DNA from the surface. For nail samples,3 mg clippings were contented in each sample and were further cut into small pieces. For both hair roots and hair stems ,3 pieces of 0.3 -0.5 cm segments were in each sample. Nuclear DNA was extracted using phenol-chloroform method, Chelex- 100 method,and kit method respectively, according to published or commercial protocols. To evaluate the reliability of the three methods for nDNA extraction, polymerase chain reaction （PCR） was conducted by using four pairs of primers located in different chromosomes with product lengths of 188 bp,248 bp,300 bp and 741 bp, respectively. PCR products were detected by 1.8% agarose gel electrophoresis. In order to determine the optimal sample quantity for each kind of material, parallel extractions with altered sample amount of 1 mg, 3 mg and 5 mg of nails, or 1 piece, 3 pieces and 5 pieces of either hair roots or hair stems were carried out and the results were summarized comparatively. A preliminary study of the eliminating effect on melanin inhibition by increasing Taq polymerase concentration was performed. Results PCR results showed that amplification of 248 bp product revealed the highest successful rate among all three kinds of extractions. For nail samples, the successful nDNA extraction rate by kit assay was significantly higher than that in phenol-chloroform and Chelex-100 methods （ P 〈 0. 05 ） ; and successful rate with phenol-chloroform method was significantly higher than that of Chelex-100 assay（ P 〈0. 05 ）. For hair stem samples, successful rate of kit assay was significantly higher than phenol-chloroform assay （P 〈0. 05 ）. However, successful rates of nDNA extraction from hair root samples using the three assays were not statistically significant （ P 〉 0. 05 ）. Among all three kinds of samples, nail sample yielded the highest successful rate （ P 〈 0.05 ） , but there was no significant difference between hair root and hair stem samples. With the sample amount increasing, successful rate of kit method increased while successful rates of phenol-chloroform and Chelex-100 methods decreased, suggesting the possibility of PCR inhibition. Conclusions （1）All three methods could be used to extract nDNA successfully from the given samples, while the kit method was recommended. （2）All three kinds of samples could be used for nDNA extraction, while free margins of finger nail could be extracted most effectively.