福尔马林固定白鱀豚标本DNA提取及其遗传多样性的初步研究

福尔马林固定标本是宝贵的遗传资源,但是如何有效利用其中的遗传信息一直存在问题。本文尝试从标本预处理、消化、PCR扩增各方面综合考虑和优化改进,成功提取并扩增21头福尔马林固定白豚标本线粒体DNA控制区410bp片段。采用了3种预处理方法尽量去除固定标本中残存的甲醛,从试验结果来看,从酒精梯度 临界点干燥处理的标本中提取的DNA在扩增时具有明显优势。通过蛋白酶K消化过程中对于酶的浓度、温浴时间的比较试验,发现随着采用大幅提高酶浓度、延长消化时间等高强度的蛋白酶消化操作后,DNA的质量和产量均得到显著提高。针对标本DNA降解严重的特点,设计特异性好且长度合适的引物以及使用巢式引物扩增,均提高了标本DNA扩增的特异性和灵敏度。通过对所测得的21头白鱀豚线粒体DNA控制区部分序列的对比,发现全部个体在该片段上的序列完全一致,说明白豚遗传多样性极低。

DNA EXTRACTION FROM FORMALIN-FIXED TISSUES AND GENETIC DIVERSITY OF BAIJI(LIPOTES VEXILLIFER)

Archival,formalin-fixed tissue is an invaluable resource for molecular genetic study,but the utilization of nucleic acid of formalin-fixed tissue is generally problematic.Because of the negative effect of formalin on proteinase K and Taq DNA polymerase,the cross-linking between proteins and DNA,DNA degradation and other reasons,in order to successfully extract and amplify DNA from formalin-fixed tissue,formalin removal,intensive protein digestion,DNA purification,and PCR optimization should all be considered.In this study,we report an integrated and modified protocol to efficiently extract and amplify DNA from baiji(Lipotes vexillifer)tissues,which were stored in unbuffered formalin for years.We used three pretreatment methods to remove formalin:gradual dehydration by alcohol,GTE buffer displacement and critical point drying,by comparison the DNA from samples treated by critical point drying is the best in quality and quantity.Intensive proteinase K treatment including increased proteinase K concentration and prolonged digestion time also greatly enhances quality and quantity of DNA.Because DNA from unbuffered formalin-fixed tissue was heavily degraded,a DNA target size of approximately 500bp or smaller was found to be optimal for PCR amplification,in addition nested PCR can greatly increase the positive results of amplification.As a result,DNA of 21 baiji specimens were successfully extracted and partial control region of mitochondrial DNA were amplified and sequenced.All the specimens have identical sequences,which indicates very low genetic diversity of baiji.