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福尔马林固定白鲟标本的DNA提取及其分子生态学初步分析
杨钟
Subtype硕士
Thesis Advisor常剑波
2007-06-06
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
Keyword白鲟 福尔马林 分子生态学 微卫星
Abstract对于白鲟这样极度濒危的物种,获取新鲜材料非常困难,本研究以中科院水生所标本馆保存的福尔马林固定白鲟标本(共11尾)为研究材料,通过一系列优化对比实验最终实现了福尔马林固定白鲟标本DNA的稳定提取,并通过相近种微卫星引物对其DNA进行了扩增,主要结论如下: 1)按DNA常规提取方法无法得到可见DNA。 2)TE9.0长时间浸泡后结合酒精梯度离心法预处理能够比较彻底地去除掉甲醛成分。 3)肌肉组织作为消化材料相比其他材料最为理想;采用组织匀浆器匀浆后消化比较彻底;提高消化液中TE浓度、PH值以及蛋白酶K、SDS的浓度并延长消化时间能够使得标本组织更容易被消化。 4)氯仿-异戊醇+饱和Nacl溶液抽提对比直接饱和Nacl溶液(6mol/L)盐析法抽提,后者得到的DNA量多,而且简单、时间耗费短。 5)沉淀DNA选用异丙醇取代无水乙醇;离心后的异丙醇重离心仍可以得到相当量的DNA。 6)试剂盒法分组对比发现酒精梯度法预处理效果不明显,两组结果差异比较显著。 7)八对匙吻鲟微卫星引物和一对中华鲟引物对从福尔马林固定白鲟组织中提取到的基因组DNA进行扩增,无法得到明显扩增条带。 8)由于从福尔马林固定标本中提取到的基因组DNA的量很少,加上含有一些复杂成分等,在分光光度计下读数会出现偏差,导致结果不准确。 9)本实验11尾个体固定时间不同(1974-1988),但所得到的DNA条带并无多大差异,从而验证了标本保存时间的长短并不影响DNA 分子的大小,而主要与保存液是否被暴露于空气中发生氧化有关。
Other AbstractIt is extremely difficult to gain the fresh specimens from Chinese Paddlefish, so this research gets the formalin-fixed tissues of Chinese Paddlefish(11 individuals) from the Institute of hydrobiology, Chinese Academy of Science. we finally get to have the DNA extracted from formalin-fixed specimens stably by optimizing a series of contrastive experiments, here are the main conclusions as follows: 1. The conventional method is unable to obtain DNA obviously. 2. the alcohol grads method and the TE9.0 for longtime soaking are better to be able to remove the formaldehyde ingredient. 3. the muscle tissues are the best of the liver, heart, muscle and fin tissues; Enhance the TE density, the PH value as well as proteinase K, the SDS density and lengthen the digestion time,these will get the tissues digested easily and protect DNA. 4. The DNA extracted by chloroform - isoamyl alcohol + saturated Nacl solution is less than by direct saturated Nacl salting-out (6mol/L) method 5. Choosing isopropyl alcohol instead of ethyl alcohol to precipitate DNA; We can also get some DNA by re-centrifuging the isopropyl alcohol. 6. The Kit method results are quite different, the effect is unstable, so it is not suitable for the samples from different years or tissues. 7. The amplifications in the different genus and family.we couldn’t get the amplification bands by using 8 pairs of microsatellite primers from Polyodon spathula and 1 pair of microsatellite primer from Chinese sturgeon. 8. Because the DNA quantity is very few, and it includes some complex ingredients, such as the metallic ions and so on, so the reading results are not correct and have the deviation under the spectrophotometer. 9. The results of the DNA extracting from the tissues of the different years (1974-1988) are nearly the same, which indicates that the time for conservation won’t affect the size of DNA, but due to whether the formalin is exposed in the air.
Pages73
Language中文
Document Type学位论文
Identifierhttp://ir.ihb.ac.cn/handle/342005/12216
Collection学位论文
Recommended Citation
GB/T 7714
杨钟. 福尔马林固定白鲟标本的DNA提取及其分子生态学初步分析[D]. 水生生物研究所. 中国科学院水生生物研究所,2007.
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