毛干DNA提取中四种细菌清除方法的比较研究

Comparative Study on the Effectiveness of Four Bacterial Removal Methods for DNA Extraction from Hair Shaft

毛发(无根)作为一种非损伤性的生物样本,在保护遗传学和分子生态学研究中发挥着十分重要的作用。以毛干中提取的DNA为模板做PCR扩增时,mtDNA片段扩增效果较好,但核基因的扩增成功率极低。人们将其归因于核DNA质低量少所致。然而我们在前期研究中发现,从毛干中得到的总DNA混有大量的细菌DNA,是导致核基因扩增困难的主要因素之一。为此我们尝试建立一种高效、快速、低成本,而且不影响DNA提取的清除毛干细菌的方法。本研究以蓝狐被毛为材料,经过普通清洗后,以4种方法处理毛干,随后使用磁珠法进行总DNA提取,并通过荧光定量PCR检测总DNA中细菌DNA的含量,以此评估4种方法的有效性。结果表明,未经处理的毛干(V#1—V#3样本)总DNA中,mtDNA和细菌pDNA的Ct值分别为(15.53±0.15)和(16.82±0.03),二者的比值为(0.923±0.009)。以SDS和NaOH(方案I)、溶菌酶(方案II)和triton X-100(方案III)处理的毛干样本,细菌DNA的含量没有明显降低。用65℃的4 mol/L NaOH溶液处理毛干样本20 s(方案IV),可把pDNA的拷贝数降低近1000倍,而动物DNA的拷贝数没有明显降低。延长处理时间至35 s以上会降低DNA的提取效果甚至提取失败。因此,我们推荐在提取毛干总DNA之前,采用方案IV处理毛干样品。

Rootless hair,as a non-invasive biological sample,plays an important role in studies of conservation genetics and molecular ecology.When total DNA extracted from hair shafts is used for PCR amplification,mitochondrial DNA(mtDNA)fragments can often be amplified,however,the success rate for nuclear genes is extremely low.This difference is attributable to the low quantity and poor quality of nuclear DNA in hair shafts.In previous research,we found that the total DNA isolated from the hair shaft contained a large amount of bacterial DNA,which was one of the main factors that caused the difficulty of nuclear gene amplification.For this reason,we tried to establish an effective,fast,and inexpensive method to remove bacteria from hair shafts without affecting DNA extraction.In this study,we tested four methods using hairs of blue fox that were first washed with laundry powder solution.Total DNA was extracted by use of the magnetic bead method,and the content of bacterial DNA and animal mtDNA in the total DNA were quantified by using real-time PCR to assess the effectiveness of the four methods.The results showed that for untreated hair shafts(samples V#1-V#3),the Ct values of mtDNA and bacterial plasmid DNA(pDNA)were 15.53±0.15 and 16.82±0.03,respectively,and their ratio was 0.923±0.009.For treatments with SDS and NaOH(Scheme I),lysozyme(Scheme II)and triton X-100(Scheme III),the bacterial DNA content did not decrease significantly.For the treatment with 4 mol/L NaOH solution at 65℃for 20 s(Scheme IV),the copy number of pDNA declined to nearly 1/1,000th of the pre-treatment level,while the copy number of animal mtDNA did not significantly decline.Prolonging the processing time to 35 s and longer dramatically reduced the effectiveness of DNA extraction or even led to extraction failure.Therefore,we recommend Scheme IV to clean hair shaft samples before DNA extraction.