短序十大功劳基因组总DNA提取方法研究

Study on DNA Extraction Method in Mahonia breviracema Y.S.Wang et Hsiao

以短序大功劳嫩叶为材料，采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA，用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率，用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明，五种方法均能从短序大功劳叶片中提取到基因组DNA，但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高，可直接用于下游分子生物学实验，CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差，不利于下游的分子生物学实验；五种方法提取的总DNA的得率在10．836-451．709μg/g之间，呈CTAB法〉SDS法〉CTAB改良法1〉CTAB改良法2〉试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。

In this paper, five different methods of DNA extraction, i.e. CTAB method, improved CTAB method 1, improved CTAB method 2, SDS method and Kit method, were used to isolate genomic DNA flom the flesh tender leaves materials of Mahonia breviracema Y.S. Wang et Hsiao. And then, the spectrophotometer, agarose gel elec- trophoresis and ISSR-PCR amplification were respectively used to investigate the genomic DNA extraction effects. The results showed that, all the five methods could extract the genomic DNA from the leaves ofM. breviracema, but there were obvious differences in extracted purity and yield of genomic DNA by with different methods. The CTAB method 2 and Kit method could yield relatively pure total DNA which were highly suited for use directly in down- stream applications. Whereas the CTAB method, improved CTAB method 1 and SDS method yield genomic DNA of poor quality which could not be used in subsequent PCR application directly. The genomic DNA yielded with five methods was ranged from 10.836 μg/g to 451.709 μg/g and was in order as the CTAB method〉SDS method〉 CTAB method 1 〉CTABCTAB method 2〉Kit method. The results could provide some basis for the molecular research of M. breviraeema.