甲醛溶液保存的中华哲水蚤基因组DNA提取和PCR扩增

Extraction of Genomic DNA and Amplification of mtDNA COI from Calanus sinicus Stored in Formaldehyde Solution

提取了保存于5%甲醛溶液中的单只中华哲水蚤基因组DNA.经凝胶电泳后虽无清晰条带,但PCR扩增后可得线粒体DNA细胞色素氧化酶I亚基基因(mtCOI)片段.测序分析,该片段长度为710bp,与GenBank中的序列(索引号AF332769)比对后确定为中华哲水蚤mtCOI序列的一部分.利用本实验方法所提取的基因组DNA可适用于系统发生、种群遗传结构等领域的分子水平研究,为保存在甲醛溶液中的小型海洋浮游动物的基因信息利用提供实用技术.

The DNA extraction,in General,from samples stored in formaldehyde solution is inefficient,especially extracted from small individual.It is necessary extracting of DNA genome from formaldehyde samples which conserved in early days or fixed in formaldehyde solution which usually adopted in marine investigation. On the base of methods of DNA extraction used in other animals or tissues,genomic DNA is extracted from Calanus sinicus stored in 5% formaldehyde solution in this experiment.More attention was paid to the process of eliminating the formaldehyde solution at the very beginning and at the digestion period,we add DTT into the digestion reaction systemwhich are the most important steps in the whole experiment.The concentration of genomic DNA in the study is low relatively,but after efficient PCR amplification,mtDNA COI gene fragment (710 bp) was obtained.Then comparing with the sequence of Calanus sinicus recorded in Genbank (accession no.AF332769),it is sure of that what we get covers the recorded of GenBank,is Calanus sinicus mtDNA COI sequence.The study will serve other small pelagic crustacean for the extracting DNA genome.It can also apply to the research of phylogeny,the population genetics and so on at molecular level,providing the use of gene information of small marine zooplankton samples stored in formaldehyde solution with practical technique.