东方白鹳粪便总DNA五种提取方法的比较

本研究比较东方白鹳(Ciconia boyciana)粪便总DNA的5种提取方法,旨在为后续物种性别鉴定和DNA条形码鉴定提供合适的方法.采用十六烷基三甲基溴化铵法(CTAB法)、十二烷基磺酸钠裂解法(SDS法)、Tiangen试剂盒法、Qiagen试剂盒法和异硫氰酸胍法(GuSCN法)对取自天津动物园的东方白鹳新鲜粪...

Methodological Comparison for Extracting Total DNA from Faeces of the Oriental White Stork (Ciconia boyciana)

[Objectives] The total DNA from fecal samples of endangered Oriental White Storks (Ciconia boyciana) was extracted in the present study by five methods (cetyltrimethylammonium bromide (CTAB) method, sodium dodecyl sulfonate (SDS) method, Tiangen kit method, Qiagen kit method, and guanidine isothiocyanate (GuSCN) method) in order to provide the suitable reference for both gender identification and DNA barcoding identification of this endangered species. [Methods] We compared the DNA concentration and purity (i.e. A260/A280 values) obtained by five extraction methods under the influence of variations in temperature which were controlled by water bath (wall breaking). Then, the mitochondrial DNA (mtDNA) D-loop sequencing which was widely used to identify and distinguish species, was selected as the target fragment for PCR amplification to test whether the obtained sequence belonged to the Oriental White Stork. The sequences were aligned by Clustal X and then used to construct a phylogenetic tree performed by Neighborhood-Joining method in MEGA 7.0, in order to identify whether the measured sequence belonged to the Oriental White Stork and analyze its evolutionary relationship with other birds. [Results] Genomic DNA could be extracted from fecal samples via each method. The DNA concentration and A260/A280 value were quite different due to the influence of water bath (wall breaking) time (Table 2). The total fecal DNA extracted by the five methods showed no obvious DNA bands in the gel electrophoresis test (Fig. 1). By comparing the DNA concentration values though Nano Drop 2000, it proved that the GuSCN method was the most efficient with the highest DNA yields reaching 90 mg/L and the purity of about 1.8 (Table 3). However, DNA yields obtained with the SDS was the lowest one. PCR analysis was performed to evaluate the quality of the extracted DNA using GuSCN method under water bath (wall breaking) time of 1 h, and the obtained specific PCR products, using D-loop primers confirmed the existence of Oriental White Storks DNA in fecal samples (Fig. 2). The sequence belonged to the Oriental White Stork from the same family gathered together from the evolutionary tree (Fig. 3), and could be distinguished from species of the same family by sequence alignment analysis (Fig. 4). [Conclusion] On the whole, the GuSCN method was proved to be the most effective one for DNA extraction from fecal samples due to its simplicity, reliability, and affordability.