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1.
福尔马林对固定标本DNA提取和扩增的影响   总被引:3,自引:0,他引:3  
夏颖哲  盛岩  陈宜瑜 《四川动物》2006,25(3):662-665
福尔马林被广泛应用于生物标本的长期保存。由于福尔马林可能影响标本DNA的质量,因此需要对福尔马林固定标本DNA的提取和扩增过程进行改进。影响从福尔马林保存标本中提取的DNA质量的主要因素包括福尔马林导致的DNA与蛋白质之间、蛋白质与蛋白质之间、DNA与DNA之间的交联,福尔马林溶液的化学成分、pH值及浓度,标本保存的时间和温度,标本保存部位等。本文总结了目前常用的对标本DNA提取和扩增过程的改进措施及其优点。  相似文献   

2.
从福尔马林保存的鱼类标本中获得高质量DNA是比较困难的。我们对前人的方法进行了如下改进:1)在标本的前处理过程中,通过长时间的缓冲液浸泡、短暂的加温、真空干燥来消除福尔马林对样品的影响;2)在样品消化过程中,加入相对过量的蛋白酶K和还原剂;3)提取DNA后立即进行PCR反应,并增加反应的循环次数和提高退火温度。通过这些改进,我们成功地从福尔马林保存的鱼类标本中提取出了高质量DNA;通过对比不同方法(福尔马林、酒精及冰冻)处理过的标本的DNA测序结果,表明该方法是值得信赖的;标本从死亡到用福尔马林处理之间的时间延搁可能是影响所提取的DNA质量的重要因素。  相似文献   

3.
若能解决馆藏陈旧标本DNA提取及PCR扩增等问题,利用模式标本或地模标本解决分类学问题将更具有说服力.本研究通过改进实验方法、优化反应条件成功地从馆藏陈旧蟹类标本中获得了靶向目的序列,并对所获序列的变异特点作了初步分析,旨在为通过改进后的方法成功获得馆藏模式标本、地模标本的目的DNA序列,以解决目前蟹类分类系统中存在的问题提供一定实验基础.  相似文献   

4.
DNA条形码主要目的是物种鉴定和新物种或隐存种的发现,而DNA条形码参考数据库是物种快速鉴定的重要基础。目前中国维管植物DNA条形码参考数据库正在建设之中,借助于公共数据库(NCBI)和初步建立的中国植物DNA条形码参考数据库,运用DNA条形码数据开展了植物标本鉴定的核查工作:(1)比较DNA序列信息与标本鉴定信息,从科、属、种级水平查找鉴定错误的标本;(2)基于有较好研究基础的DNA条形码参考数据库,开展未知标本的鉴定;(3)通过对标本核查的总结,提出DNA条形码参考数据库建设过程中的几点建议。  相似文献   

5.
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头福尔马林固定白暨豚标本线粒体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2l头白暨豚线粒体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,说明白暨豚遗传多样性极低。  相似文献   

6.
陈旧皮张中DNA提取的新方法   总被引:26,自引:6,他引:26       下载免费PDF全文
对传统的馆藏陈旧皮张标本DNA提取方法进行了改进,所提DNA分子量可达1kb,而且具有样品用量少(约0.01g),消化时间短(约14h)和操作步骤简单等优点,利用所提DNA,对小熊猫等珍稀动物线粒体DNA的细胞色素b和控制区序列的部分片段进行了PCR扩增,序列测定和比较分析,证实所提DNA合格而无污染,完全可以用于珍稀动物保护遗传学研究。  相似文献   

7.
不同保藏处理的昆虫标本DNA提取及其随机扩增多态DNA反应   总被引:15,自引:0,他引:15  
实验利用CTAB法对柳二十斑叶甲Chrysomelavigintipunctata (Scopoli)、异色瓢虫HarmoniaaxyridisPollas、七星瓢虫Coc cinellaseptempunctataLinnaeus、小地老虎Agrotisypsilon (Rottemberg)、红蜻CrocothemisserviliaDrury、无齿稻蝗OxyaabentataWil lemse和中华稻蝗Oxyachinensis (Thunberg)等 7种昆虫进行了基因组DNA提取。从自然干燥标本、烘干标本及酒精浸泡标本获得的DNA均可用于RAPD PCR反应 ,且烘干标本、酒精浸泡标本提取效果优于自然干燥标本。这种提取方法简便易行 ,容易掌握 ,且耗资小于其它分子生物学方法。  相似文献   

8.
不同保藏处理的昆虫标本DNA提取及其随机扩增多态DNA反应   总被引:19,自引:0,他引:19  
张迎春  刘波等 《昆虫学报》2002,45(5):693-695
实验利用CTAB法对柳二十斑叶甲Chrysomela vigintipunctata (Scopoli)、异色瓢虫Harmonia axyridis Pollas、七星瓢虫Coccinella septempunctata Linnaeus、小地老虎Agrotis ypsilon (Rottemberg)、红蜻Crocothemis servilia Drury、无齿稻蝗Oxya abentata Willemse和中华稻蝗Oxya chinensis (Thunberg)等7种昆虫进行了基因组DNA提取。从自然干燥标本、烘干标本及酒精浸泡标本获得的DNA均可用于RAPD-PCR反应,且烘干标本、酒精浸泡标本提取效果优于自然干燥标本。这种提取方法简便易行,容易掌握,且耗资小于其它分子生物学方法。  相似文献   

9.
甲螨是一类重要的土壤动物,体型微小,一般具有较厚的体壁。本研究针对甲螨这一特定类群,探讨了一种无形态特征损伤的DNA提取技术。通过结合试剂盒DNA提取法,并适当改进实验条件,设计出一套行之有效的DNA提取流程。通过对提取DNA之后的标本进行形态学观察,发现其主要的分类学特征均保存完好,可以作为凭证标本长期保存。本研究所提供的DNA提取技术既可以提取出足够的DNA又可以保留凭证标本,因此能有效促进甲螨分子分类学相关研究。  相似文献   

10.
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。  相似文献   

11.
李楠 《人类学学报》2019,38(1):98-106
作为普通生命表的归纳总结,“模型生命表”反映了人口发展的普遍现象和内在规律。本文将“区域模型生命表”引入古人口学研究,以大甸子墓地为例对婴幼儿组和高龄组死亡人数进行了调整。根据校正后数据所编制的简略生命表,该遗址人口平均预期寿命下降为24.12岁,年龄别死亡率曲线呈更加合理的“U”型。鉴于古人口学中样本容量和代表性往往较差,引入“区域模型生命表”对偏差数据校正后再进行人口研究将使所得结论更加合理可靠。  相似文献   

12.
动物食性分析是动物营养生态学的重要研究手段,可用于解析动物与环境因素的关联性、捕食者与猎物之间的关系,以及动物物种多样性等科学问题。近年来,基于新一代测序技术的DNA宏条形码技术被广泛应用到生态学多个研究领域,极大地促进了生命科学交叉学科的发展。其中,DNA宏条形码技术在动物食性分析中具有高分辨、高效率、低样本量等优势,具有重要的应用前景。综述了基于DNA宏条形码技术的动物食性分析在生态学中的应用研究进展,并进一步总结了DNA宏条形码技术原理和食性分析方法,着重探讨了基于DNA宏条形码技术的动物食性分析在珍稀濒危动物保护、生物多样性监测、农业害虫防治等生态学研究领域中的应用,并对DNA宏条形码技术在动物食性分析中存在的问题及应用前景进行小结与展望。  相似文献   

13.
Maintaining genetic diversity is a crucial component in conserving threatened species. For the iconic Australian koala, there is little genetic information on wild populations that is not either skewed by biased sampling methods (e.g., sampling effort skewed toward urban areas) or of limited usefulness due to low numbers of microsatellites used. The ability to genotype DNA extracted from koala scats using next‐generation sequencing technology will not only help resolve location sample bias but also improve the accuracy and scope of genetic analyses (e.g., neutral vs. adaptive genetic diversity, inbreeding, and effective population size). Here, we present the successful SNP genotyping (1272 SNP loci) of koala DNA extracted from scat, using a proprietary DArTseq? protocol. We compare genotype results from two‐day‐old scat DNA and 14‐day‐old scat DNA to a blood DNA template, to test accuracy of scat genotyping. We find that DNA from fresher scat results in fewer loci with missing information than DNA from older scat; however, 14‐day‐old scat can still provide useful genetic information, depending on the research question. We also find that a subset of 209 conserved loci can accurately identify individual koalas, even from older scat samples. In addition, we find that DNA sequences identified from scat samples through the DArTseq? process can provide genetic identification of koala diet species, bacterial and viral pathogens, and parasitic organisms.  相似文献   

14.
We describe a simple and efficient method for genomic DNA extraction from woody fruit crops containing high polysaccharide levels. This method involves a modified CTAB or SDS procedure employing a purification step to remove polysaccharides by using water-saturated ether and 1.25 M NaCl. Precipitation with an equal volume of isopropanol caused a DNA pellet to form. After being washed with 70% ethyl alcohol, the pellet easily dissolved in TE buffer. Using this method, DNA was extracted from samples of more than 1000Citrus spp., including young leaves, old leaves, frosted old leaves, withered old leaves, and callus lines. The average yield of DNA ranged from 50–500 μg/g of sample. DNA was suitable for PCR and RFLP analyses and long-term storage. Recently, the procedure was used to isolate DNA from withered old leaves of more than 20 tropical and subtropical fruit crops.  相似文献   

15.
Abstract: Neurons do not divide during adult life and thus they provide a unique system to study the effects of age-accumulated damage to DNA in the absence of DNA replication. We have analyzed DNA polymerase activity in neurons isolated from young adult and very aged mice. The predominant catalytic activity is DNA polymerase-β and it is present in similar amounts in neurons from young and old mice. This polymerase is highly errorprone in copying φX174 DNA, the error frequency being about 1/7,000 and not significantly different when obtained from young and old animals. This high infidelity is considered with respect to DNA repair and the protein synthesis error catastrophe theory of aging.  相似文献   

16.
17.
—The levels of DNA, RNA, protein and activities of acid and alkaline DNases in developing and old chicken brain were studied. A rapid increase in DNA content was found in the embryonic brain until just prior to hatching. Thereafter, with a transient plateau around the day of hatching, the DNA continued to increase but at a very slow rate. Two-year-old brain was found to have a markedly higher level of DNA as compared to the 10th day postnatal value. RNA showed a steady increase up to the 20th day of embryonic life. Protein levels showed a gradual increase throughout the period studied. Both acid and alkaline DNases exhibited maximum activity during embryonic life, i.e. at a time when rapid cellular proliferation was occurring. With advancing age, the acid DNase activity showed a marked decline thus exhibiting no correlation to the high level of DNA found in the old brain. The alkaline DNase activity, however, was still at a significant level in the 2-year-old brain.  相似文献   

18.
19.
The prevalence of spontaneous mutations increases with age in the male germline; consequently, older men have an increased risk of siring children with genetic disease due to de novo mutations. The lacI transgenic mouse can be used to study paternal age effects, and in this system, the prevalence of de novo mutations increases in the male germline at old ages. Mutagenesis is linked with DNA repair capacity, and base excision repair (BER), which can ameliorate spontaneous DNA damage, decreases in nuclear extracts of spermatogenic cells from old mice. Mice heterozygous for a null allele of the Apex1 gene, which encodes apurinic/apyrimidinic endonuclease I (APEN), an essential BER enzyme, display an accelerated increase in spontaneous germline mutagenesis early in life. Here, the consequences of lifelong reduction of APEN on genetic instability in the male germline were examined, for the first time, at middle and old ages. Mutant frequency increased earlier in spermatogenic cells from Apex1(+/-) mice (by 6 months of age). Nuclear DNA damage increased with age in the spermatogenic lineage for both wild-type and Apex1(+/-) mice. By old age, mutant frequencies were similar for wild-type and APEN-deficient mice. Mitochondrial genome repair also depends on APEN, and novel analysis of mitochondrial DNA (mtDNA) damage revealed an increase in the Apex1(+/-) spermatogenic cells by middle age. Thus, Apex1 heterozygosity results in accelerated damage to mtDNA and spontaneous mutagenesis, consistent with an essential role for APEN in maintaining nuclear and mtDNA integrity in spermatogenic cells throughout life.  相似文献   

20.
Isolation of high-quality DNA from plants, especially plants from the Cerrado, is notoriously difficult because of polysaccharides and secondary compounds produced by plants from this biome. DNA isolation and its quality may be compromised by chemical defenses such as tannins and phenols. Quantitative plant defenses tend to have a cumulative effect, increasing in concentration during leaf development, reducing DNA quality extracted in mature compared to young leaves. We report the effect of leaf age on DNA extraction of Dimorphandra mollis. Our working hypothesis was that the young leaves have more DNA than old leaves of the same individual because chemical defenses accumulate in older leaves. Young and old leaves were sampled from eight mature trees as well as leaves from eight seedlings in the north region of Minas Gerais State. Genomic DNA extraction followed the standard CTAB procedure. DNA isolation was very successful from young leaves of 16 individuals of D. mollis. The extracted DNA exhibited high quality and the DNA quantity was also high, with an A(260)/A(280) ratio above 1.8, which is within the optimal sample range. In contrast, DNA isolation from old leaves was not successful. When the DNA was extracted from old leaves, the DNA was brownish, indicating contamination by phenolic compounds. These metabolites oxidize the DNA irreversibly, which hinders amplification of DNA by PCR by inhibiting the action of enzymes such as Taq polymerase. PCR performed with DNA from young leaves of D. mollis was successful and produced strong bands for RAPD markers.  相似文献   

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