乙醇保存的动物标本基因组DNA提取方法的比较

为提高从乙醇长期保存的动物标本中提取大分子DNA的质量,用5种不同方法对动物组织进行预处理实验,然后采用SDS／蛋白酶K裂解,酚一氯仿抽提和乙醇沉淀提取总DNA,通过0．8％琼脂糖凝胶对模板进行电泳和PCR产物作鉴定,经比较,用0．9％NaCL法、PBS法和混合液法进行预处理,消除乙醇对Taq酶的影响以及蛋白质和核酸交联问题,为提取动物基因组DNA的3种更理想方法。     

鳞翅目昆虫基因组中微卫星DNA的特征以及对其分离的影响

本文根据我们对鳞翅目昆虫棉铃虫和松毛虫以及其它动物 (筏蜘蛛、朱、鳕鱼和飞蝗 )的微卫星富集性基因组DNA文库的筛选和分析结果 ,结合其它实验室已发表的资料 ,对鳞翅目昆虫基因组中微卫星DNA的丰度和结构特点进行了较为系统的分析。结果表明 :与其它类群相比 ,尽管鳞翅目昆虫物种间存在差异 ,但其基因组中存在明显偏多的侧翼序列重复的、以多拷贝形式存在的微卫星位点 ,且其中相当一部分以基因家族的形式存在。微卫星DNA家族通常可以在序列分析阶段被识别出来 ,但很多多拷贝位点只有通过一系列后续分析才能被检查出来。这应是鳞翅目昆虫中微卫星位点的优化率相对偏低的主要原因。棉铃虫和松毛虫基因组中三相重复微卫星丰度相对较高 ,从而从某种程度上补偿了这些物种微卫星分离过程中因丰度低、多拷贝位点比例高所带来的困难。棉铃虫微卫星DNA家族侧翼序列中多聚T/A序列的存在表明 ,逆转录转座或逆转录侵染可能是在基因组中形成多拷贝微卫星位点和微卫星DNA家族的重要机制之一     

松墨天牛成虫标本保存及其DNA提取质量比较

【目的】探讨适合DNA提取的天牛成虫标本保存方法。【方法】采用SDS-蛋白酶K消化法对液氮中冷冻保存、无水乙醇-20℃冷冻保存、无水乙醇室温保存和干标本室温保存且保存时间在2年以上的松墨天牛Monochamus alternates Hope成虫标本基因组DNA进行提取,并对不同保存方式提取的DNA样本进行了质量比较和分析。【结果】在上述常见的松墨天牛成虫标本4种保存方式中,以液氮中冷冻保存效果最佳,其次为无水乙醇-20℃冷冻保存,插针干标本室温保藏效果最差。利用昆虫线粒体基因CO I和CO II的通用引物从上述DNA中均能够成功扩增出目的片段,测序结果证实扩增片段符合预期。【结论】液氮和无水乙醇-20℃冷冻保存适合松墨天牛成虫标本长期保存,且不影响后续的PCR扩增和测序。     

福尔马林保存的动物标本基因组DNA的提取方法

从在福尔马林长期保存的动物标本中提取基因组DNA用于分子生物学研究一直是一个未得到彻底解决的难题。本文提出了一种从浸泡在福尔马林的大仓鼠肝脏和日本鳗鲡肌肉标本中提取基因组DNA的新方法。取浸泡于福尔马林中的大仓鼠肝脏或日本鳗2鲡肌肉适量,用PBS溶液冲洗,放在灭菌的吸水纸上将其揩干,于超净工作台内用无菌剪刀将材料剪成50mg的小块,放入PBS液浸泡12－24h;然后转入70％的乙醇中处理12－24h。依次换入下列梯度酒精中处理：80％乙醇,2h,重复一次;90％乙醇,2h,重复一次;95％乙醇,2h,重复一次;100％乙醇,1h,重复一次。然后将材料放入1／2倍的PBS液中浸泡12h。其间更换一次溶液。提取方法参考Sambroock等人（1989）,加蛋白K瓣量按标准量（100μg/mlL),在50－56℃温浴3－6h处理过程中,不断轻摇混匀,视消化效果可以重复加入标准量的1／2倍蛋白酶K进行消化,直至将材料完全消化为止;酚氯仿抽提最后一次的上清液移入透析袋中透析;沉淀DNA时,在－20℃下20min效果为宜。该方法主要特点在于对标本进行预处理,在保证不使DNA进一步了解的前提下,首先去除标本中所含的福尔马林溶液的成分,然后利用改进的酚氯仿抽提法提取该类标本的基因组DNA,再进一步用透析法纯化DNA。研究结果表明,采用该方法提取和纯化被试标本基因组DNA能较好地应用于RAPD,微卫星位点的PCR扩增、Suthern和斑点杂交。     

DNA条形码在鳞翅目昆虫中的应用

2003年,Hebert等提出DNA条形码后,快速而精确的特点使它在物种鉴定中得到了广泛的应用。鳞翅目是昆虫纲中第二大目,其物种鉴定任务复杂而艰巨,因此DNA条形码具有广阔的应用前景。该文主要针对DNA条形码概况以及近年来它在鳞翅目昆虫中的研究情况予以综述。     

微卫星DNA在吉戎兔亲子鉴定中的应用研究

选用Sat2、Sat3、Sat4、Sat5、Sat7、Sat8、Sat12、Sat13、Sat16、Sol08、Sol28、Sol30、Sol03 等13个微卫星位点PCR扩增30只吉戎兔的基因组DNA,然后在8%聚丙烯酰胺凝胶上电泳分型,检测结果为平均等位基因数为3.46个,平均杂合度（H）为0.578,平均多态信息含量（PIC）为0.531,双亲资料未知时13个位点的累计非父排除率（PE1）为0.935226,置信度低于80%,一亲本资料已知时13个位点的累计非父排除率（PE2）为0.999329,置信度为95%。由于所鉴定兔群的母本资料已知,因此基因型的分型结果能有效确认18只仔兔子的真父。     

膜翅目昆虫干标本的基因组DNA提取

提出了膜翅目昆虫干标本基因组DNA的提取方法,通过对实验结果和所测得的基因片段（28S D2 rDNA)的分析,表明该方法可行。     

不同保存条件下茧蜂标本基因组DNA的降解

探讨了不同保存条件下高温、氧化、化学修饰等对茧蜂标本基因组DNA降解的影响,提出用含50 mmol.L-1乙二胺四乙酸的无水乙醇保存室内寄生蜂标本,并在液面覆盖石蜡油等可行性建议。     

麦冬基因组DNA提取方法研究

为了从富含多糖、多酚的顽拗植物麦冬中分离出高质量基因组DNA,分别建立了改良CTAB法和改良SDS法。通过使用核分离液和CTAB/NaCl溶液、提取液中加入0.35倍体积无水乙醇等最大限度地除去杂质。将两种方法提取的8种麦冬DNA与两种离心柱式试剂盒提取的DNA在纯度、产率等方面进行对比,并进行双酶切和SRAP分析。麦冬类植物SRAP反应体系的建立尚属首次。结果表明,改良CTAB法和改良SDS法均能从8种麦冬叶片中提取到高质量的基因组DNA,改良CTAB法提取纯度更高,提取幼叶DNA纯度可以与离心柱式试剂盒媲美,能够满足多种分子生物学试验要求。在后续试验中,用改良CTAB法从20多种沿阶草族植物中提取到了高纯度DNA。该方法通用性好,提取的DNA纯度高,对其他顽拗类植物基因组DNA的提取具有借鉴意义。     

微卫星DNA标记技术及其在遗传多样性研究中的应用

微卫星DNA的高突变率、中性、共湿性及其在真核基因组中的普遍性,使其成为居群遗传学研究、种质资源鉴定、亲缘关系分析和图谱构建的优越的分子标记。本研究系统介绍了微卫星DNA在结构和功能上的特点,并对微卫星DNA标记技术应用的遗传学机理和一般方法进行了扼要的阐述。另外,本研究还探讨了微卫星DNA标记技术在遗传多样性研究中的应用现状,并进一步提出其发展前景。     

Factors affecting DNA preservation from museum-collected lepidopteran specimens

Recent innovations in molecular genetics made DNA an intriguing molecule not only in molecular biology, but also in ecology and evolutionary and conservation biology. Despite this general interest, several discrepancies have been reported in the literature regarding the techniques for preserving insects for DNA analysis, prompting us to analyse the effects of different storage conditions on lepidopteran DNA preservation. In particular, in the present paper, adults of the cabbage moth, Mamestra brassicae (L.) (Lepidoptera: Noctuidae), were stored under various conditions in order to verify which method is the most suitable to preserve lepidopteran specimens for DNA studies. Mamestra brassicae adults were stored by rapid desiccation with silica gel, by preservation in acetone, 2-propanol, Carnoy's or ethanol (both at 75 and 100% concentrations) solutions, and finally by storage in an ultracold freezer and liquid nitrogen. Adults preserved by each method were used to extract DNA at the aim of verifying the size of the extracted DNAs, the extraction yield and the possibility of using these samples to amplify both short and long DNA sequences by polymerase chain reaction. The results were compared with those obtained using fresh samples acting as controls. Acetone preservation appeared to be the most recommendable method for moth specimens as it proved to be a good storage medium for DNA analysis, it is cost-effective, and it is applicable not only to field surveys, but also to obtain efficient and low-cost storage of lepidopteran specimens in museum collections.     

东方粘虫(Pseudaletia separata(Walker))微卫星富集文库的构建与分析

根据生物素与链亲和素的强亲和性原理,用包被链亲和素的磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)15、(GA)15、(GTT)12、(GAT)12、(TAGA)8 和 (GTGA)8退火结合的含接头和微卫星序列的单链粘虫基因组DNA限制性酶切片段,获得单链目的片段.经PCR扩增形成双链后连接到pGEM-T 载体上,再转化到DH5α热感受态细胞中,首次成功构建粘虫基因组微卫星富集文库.随机抽样(16次抽样,每库共12～114个克隆)测序发现,6个文库总的微卫星阳性克隆率30.07%,CA/GAT/GTT 等3个文库的阳性克隆率达到40%以上.单次抽样最高阳性克隆率达到56%.粘虫微卫星富集文库的建立和高多态性微卫星单拷贝位点的筛选将为粘虫的生态遗传学研究、连锁图谱构建、分子进化和系统发育研究等提供大量遗传标记,对昆虫迁飞的分子生态研究也有重大意义.     

蚜虫基因组DNA提取方法的改进

蚜虫基因组DNA的提取是蚜虫分子生物学研究中的难点。参照动物基因组DNA的提取方法,根据蚜虫体型微小,体表有外骨骼的特点,对SDS法作了改进。改进的方法无需用组织捣碎棒破碎虫体,操作简便。与现在常用的提取方法相比,改进的SDS法能快速、有效地提取单头蚜虫的基因组DNA,适用于RAPD随机引物和测序引物的PCR扩增。     

Ultra-simple DNA extraction method for marker-assisted selection using microsatellite markers in rice

We prevent an ultra-simple DNA extraction method for microsatellite analysis of rice. Each extraction requires only one microtube, one disposable pipette tip, TE buffer and few pieces (about 5 mm) of rice leaf tissue. This is sufficient for 200 PCR reactions. The extract can be kept in the freezer for long-term storage. Also, DNA can be extracted from 200–300 individuals in a few hours. These features enabled us to perform rapid largescale seedling genotyping required for marker-assisted selection. We have also examined the applicability of this method for other PCR-based markers: RAPDs, nuclear STS, chloroplast STS and chloroplast microsatellites.     

Cross-amplification of microsatellite loci in a neotropicalQuercus species and standardization of DNA extraction from mature leaves dried in silica gel

Conservation of 15 out of 24 previously identified microsatellite loci (62.5%), was found in a survey of the South American oak,Quercus humboldtii. The number of alleles per locus varied from 2 to 20, detecting at least 5 microsatellite loci with 5 or more alleles. This number of loci and alleles is adequate for most studies of genetic diversity and gene flow analysis. In addition, a method for extracting DNA from mature oak leaves is described that minimizes oxidation of tannins, a common problem in silica-gel-dried samples. The microsatellite markers detected in this study and the DNA extraction protocol may be applied to more than 30 species ofQuercus that exist in tropical America.     

Development of 51 genomic microsatellite DNA markers of guppy (Poecilia reticulata) and their application in closely related species

Fifty‐one microsatellite DNA markers of guppy (Poecilia reticulata) were developed. All of the markers detected moderate allelic variation. The number of alleles for each locus ranged from two to 10. Observed and expected heterozygosities varied from 0.10 to 0.63 and from 0.23 to 0.77, respectively. Three additional fish species of family Poeciliidae were used for the cross‐species amplification of these markers. It was found that only four to 16 markers detected polymorphism in these three fish species.     

Rapid and efficient extraction of genomic DNA from differentphytopathogenic fungi using DNAzol reagent

A modified procedure using the commercial DNAzol reagent was successfully applied to extract genomic DNA from 25 fungal species. The DNA yield varied from 306 to 1,927 g g^-1 dry mycelia and the A₂₆₀/A₂₈₀ ratio from 1.59 to 1.93. Compared with the method of J.L. Cenis (Nucleic Acids Res. 1992, 20: 2380) this procedure generated a higher DNA yield from 17 species and a higher A₂₆₀/A₂₈₀ ratio from 23 species. But for four species, Cenis (1992) method was more suitable. No inhibitor of polymerase chain reaction was evident for the DNA extracted by the modified procedure, whereas some inhibitors remained in DNA of eight species extracted by the previous method.Revisions requested 8 September 2004; Revisions received 1 November 2004     

苔藓植物DNA提取方法研究

提取高质量的 DNA是对苔藓植物遗传多样性进行研究的基础。该文以苔藓植物为试材 ,用 5种方法 ,即快速提取法、改良 CTAB法、CTAB法、SDS法及高盐法 (第一种为自行设计 ,第二种是对原有方法的改进 )对苔藓植物 DNA提取方法进行了比较研究。结果表明 ,快速提取法和改良 CTAB法是 2种适合于苔藓植物 DNA提取的方法。这 2种方法提取的 DNA浓度和纯度均比较高 ,凝胶电泳显示无明显降解现象 ,适宜作为 PCR扩增的模板 ,并成功地进行了 RAPD扩增。