藏酋猴微卫星富集文库的构建及微卫星分子标记的筛选

通过磁珠富集法构建了藏酋猴AC重复和AAAG重复的微卫星富集文库,分离微卫星序列并对其进行分析。将藏酋猴基因组DNA经Sau3AI酶切后纯化回收,连接特定接头。用生物素标记的探针与酶切片段杂交,捕获300～1000bp片段,随后将获得的片段连接到pMD-19T载体上,转化至JM109中,成功构建藏酋猴微卫星富集文库。(AC)n富集文库和(AAAG)n富集文库的阳性克隆率分别为50%和10%左右。根据测序得到的48个微卫星序列成功设计了24对引物,最终筛选出6个微卫星标记,这些标记将为藏酋猴的遗传多样性研究、圈养种群结构的分析和遗传图谱的构建等奠定一定的基础。     

磁珠富集法筛选实验豚鼠微卫星分子标记

目的直接从实验豚鼠基因组DNA中筛选获得微卫星分子标记。方法应用磁珠和生物素标记的微卫星探针与豚鼠基因组酶切片段杂交,捕获200～1000 bp含有微卫星序列的DNA片段,连接到pMD-18V载体中,转化到感受态细胞E.coli DH5α中构建富集微卫星序列的小片段插入文库。然后用PCR法进行筛选。结果从约2000个转化子中获得240个阳性克隆。对其中98个进行了测序,并成功设计豚鼠微卫星引物17对。结论经过优化的磁珠富集法能够稳定、高效地获得豚鼠微卫星标记。本研究获得的微卫星位点将成为豚鼠遗传学研究的有力工具。     

云南松基因组微卫星富集文库的构建

采用磁珠富集法构建云南松微卫星富集文库。云南松基因组DNA经RsaⅠ酶切,与特定接头连接,再用接头特异引物进行PCR扩增。连接扩增产物与用生物素标记的(AG)12、(AT)12、(CG)12、(GT)12、(ACG)12、(ACT)12和(CCA)8探针杂交,通过链霉亲和素偶联的磁珠捕捉含接头和微卫星序列的片段并扩增,将获得的片段连接到pMD-19T载体上,转化至大肠杆菌JM109感受态细胞中,成功构建了云南松微卫星富集文库。通过PCR检测从文库中筛选阳性克隆,在383富集阳性菌落中获得阳性克隆257个,经测序分析,在获得的159条序列中,有143条含有SSR,其中完美型占65.73%,非完美型占23.78%,混合型占10.49%。结果表明,磁珠富集法构建云南松基因组微卫星文库高效、可行的,文库的构建为微卫星位点的分离、遗传多样性的分析等奠定基础。     

Dynal磁珠富集大熊猫微卫星标记

应用Dynal磁珠-生物素标记的微卫星探针与大熊猫基因组酶切片段杂交,捕获400—600bp含有微卫星序列的DNA片段,连接到pGEM-T载体中,构建富集微卫星序列的小片段插入文库。应用γ^32P标记的探针筛选文库,从2880个转化子中获得了260个阳性克隆。对54个序列进行了测序,并成功地设计了大熊猫微卫星引物37对。该方法能有效提高筛选微卫星标记的效率。     

背角无齿蚌基因组(GT)n微卫星DNA特征

利用磁珠富集法筛选背角无齿蚌(Anodonta woodiana)的微卫星分子标记,采用Sau3A1酶对完整DNA进行酶切,以生物素标记的(GT)15寡核苷酸探针从酶切片段中筛选微卫星序列.洗脱的杂交片段克隆到PGEM-T载体上构建富集微卫星基因组文库后,通过菌液PCR筛选检测出阳性克隆进行测序.结果表明:在筛选的18...     

磁珠富集法分离刀鲚微卫星标记

利用磁珠富集法分离微卫星序列,以开发长江刀鲚微卫星分子标记。将长江刀鲚基因组DNA经限制内切酶Mse I酶切,回收400-1 000 bp片段,安装接头,构建长江刀鲚全基因组PCR文库。用生物素标记的微卫星探针(CA)12与其杂交,磁珠富集含有微卫星序列的DNA片段。将洗脱所得片段进行PCR扩增,然后进行克隆。经过菌落PCR检验后挑选出118个阳性克隆进行测序,其中97条含有微卫星序列。用设计合成59对微卫星引物对30尾养殖长江刀鲚进行引物的多态性筛选,得到9对多态性引物。     

牦牛基因组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,用链亲和素磁珠亲和捕捉与生物素标记的微卫星寡核苷酸探针(CA)12、(CCG)8、(CAG)8、(TTTC)8退火结合的含有接头和牦牛微卫星序列的单链限制性酶切片段,获得单链目的片段,经PCR扩增形成双链,然后克隆到pMD18-T载体上,转化至DH5α中,首次成功构建牦牛基因组微卫星富集文库。测序结果发现,阳性克隆率为77％(37／48),说明构建的牦牛基因组微卫星富集文库是一个高质量的文库。牦牛富集微卫星文库的建立和牦牛微卫星的筛选将为下一步进行牦牛基因组结构的分析、牦牛遗传连锁图谱的构建、分子进化和系统发育研究、标记辅助选择以及经济性状的QTL定位提供大量的微卫星标记。     

四川梅花鹿(CAG)n微卫星文库构建

应用Dynal磁珠-生物素标记的微卫星探针与四川梅花鹿基因组酶切片段杂交,捕获200～750 bp含有微卫星序列的DNA片段,连接pMD18-T载体,再转化到感受态细胞JM109中以构建文库.通过PCR方法从(CAG)n文库中筛选阳性克隆,从576个转化子中获得了234个阳性克隆,对其全部进行序列测定,其中73个含有微卫星序列.除获得的目的微卫星序列(CAG)n外,还观察到(AG)n、(AT)n的重复序列.本研究表明,经过优化的磁珠富集法能够稳定、高效地获得四川梅花鹿微卫星标记.     

曼氏无针乌贼GT微卫星位点的筛选

采用链霉亲和素包被的磁珠富集法筛选曼氏无针乌贼(Sepiella maindroni)微卫星位点。试验样品来自舟山六横岛,提取4个样品的DNA混合成DNA pool,用限制性内切酶Sau 3A I酶切。接上接头后构建基因组PCR文库,用生物素标记的(GT)15探针筛选。将筛选获得目的片段进行PCR扩增,连接pMD18-T载体,转入DH5α感受态大肠杆菌里,扩大培养后PCR筛选阳性克隆。总共选取278个克隆,对120个经过检测含有插入片段的克隆进行测序,发现102个克隆含有微卫星序列,阳性克隆比率为85%。除去重复测序和侧翼链不足的序列,可以设计引物的微卫星序列有64条。     

细梢小卷蛾微卫星富集文库的构建与分析

目的:构建细梢小卷蛾Rhyacionia leptotubula微卫星富集文库.方法:提取细梢小卷蛾基因组DNA,经限制性内切酶Rsa Ⅰ酶切,用(CT)10和(GT)10生物素探针与其杂交,利用磁珠富集含有微卫星的DNA序列,并对其进行PCR扩增,将扩增产物连接到pMD18-T载体后转入感受态大肠杆菌DH5α中,得到微卫星富集文库:结果:对100个克隆进行随机测序,获得98个微卫星序列,其中具有5次及以上碱基重复次数的微卫星克隆占26%,最高碱基重复次数为33次,非完美型占12%,说明构建的细梢小卷蛾微卫星富集文库是一个高质量的文库.结论:该文库的建立为后续筛选具高多态性的微卫星标记引物研究细梢小卷蛾的种群遗传结构、迁移扩散规律等奠定了基础.     

Microsatellite instability.

Unlike aneuploidy, considered to be the cardinal feature of malignant tumors ever since the chromosomal analysis of neoplastic cells became technically feasible, a second pathway toward malignancy has emerged over the past decade that is not characterized by gross aneuploidy but, instead, by inactivation of the DNA mismatch repair system, leading to a hypermutable state in which simple repetitive DNA sequences are unstable during DNA replication. Although mutations of many of these microsatellite sequences are presumably innocuous, because they do not occur in the coding or regulatory regions of genes, other such sequences are critically located in the coding regions of genes involved in the regulation of cell growth. First discovered in the rather uncommon hereditary nonpolyposis colorectal cancer syndrome, where there is an inactivating germline mutation in one of the DNA mismatch repair genes and most of the tumors show microsatellite instability, the latter phenomenon has since been implicated in about 15% of sporadic colorectal cancers, as well as in cancers at several other sites, such as the endometrium. Tumors showing microsatellite instability are generally near-diploid, are at a low stage of development, have a favorable prognosis, and, in the colon, are commonly located on the right side. In recent years, epigenetic phenomena, including hypermethylation and loss of imprinting, have come to be recognized as having a significant bearing on the development of these tumors.     

Microsatellite Instability in Yeast: Dependence on the Length of the Microsatellite

One of the most common microsatellites in eukaryotes consists of tandem arrays [usually 15-50 base pairs (bp) in length] of the dinucleotide GT. We examined the rates of instability for poly GT tracts of 15, 33, 51, 99 and 105 bp in wild-type and mismatch repair-deficient strains of Saccharomyces cerevisiae. Rates of instability increased more than two orders of magnitude as tracts increased in size from 15 to 99 bp in both wild-type and msh2 strains. The types of alterations observed in long and short tracts in wild-type strains were different in two ways. First, tracts >/=51 bp had significantly more large deletions than tracts </=33 bp. Second, for the 99- and 105-bp tracts, almost all events involving single repeats were additions; for the smaller tracts, both additions and deletions of single repeats were common.     

微卫星标记及其在鸟类中的应用

微卫星DNA作为一种分子标记,以其诸多优点被认为是各类遗传标记中最有价值的一种。目前已被广泛应用于鸟类的研究中。本文介绍了微卫星DNA标记的分布特点、突变机制和作为分子标记的特点,并综述了微卫星DNA标记在鸟类的亲权分析及种类鉴定、遗传图谱构建及基因定位、物种进化历史揭示和遗传多样性研究中应用现状。     

Microsatellite genotyping of carnation varieties

A set of 11 sequence-tagged microsatellite markers for carnation (Dianthus caryophyllus) was developed using a DNA library enriched for microsatellites. Supplemented with three markers derived from sequence database entries, these were used to genotype carnation varieties using a semi-automated fluorescence-based approach. In a set of 82 cultivars, the markers amplified 4-16 alleles each. The effective number of alleles varied from 1.9 to 6.0. For the eight best scorable markers, heterozygosity was between 0.51 and 0.99. The markers were able to distinguish all cultivars with a unique combination of alleles, except for sport mutants, which were readily grouped together with the original cultivar. In addition, one group of three and one group of six cultivars each had the same combination of 'allelic peaks'. The cluster of three varieties concerned original cultivars and their mutants. The cluster of six consisted of four mutants from the same cultivar and two other varieties.     

微卫星方法简介

８０年代以来，分子生物学技术与方法已成为进化生物学、保护遗传学、生态学等诸多领域非常重要和崭新的研究工具。而摆在人们面前可供选择的分子生物学方法又有许多种，如同工酶（ａｌｏｚｙｍｅｓ）、限制性片断长度多态性（ＲｉｓｔｒｉｃｔｉｏｎＦｒａｇｍｅｎｔＬｅ...     

Microsatellite DNA in fishes

For the last 30 years, attempts have been made to discriminate among fish populations by using molecular markers. Although some techniques have proved successful in certain circumstances, the consistent trend to newer markers among fishery geneticists highlights the general lack of resolving power observed with older technologies. The last decade has seen the increasing use of satellite DNA in investigations of genetic variability and divergence. Applications to fish and fisheries-related issues initially concentrated on minisatellite single-locus probes. Although minisatellites have successfully addressed a number of fishery-related questions, this class of satellite DNA has not been widely adopted by fishery geneticists. Most of the current research effort is concentrated on another class of satellite DNA called microsatellites. The large interest in microsatellite loci is largely due to the very high levels of variability that have been observed and the ability to investigate this variation using PCR technology. The isolation and application of microsatellites to research fields as diverse as population genetics, parentage analyses and genome mapping are reviewed. Despite the undisputed advantages that the marker possesses, there are a number of potential problems associated with investigating variation at microsatellite loci. Statistical considerations (e.g. appropriate sample sizes, number of loci and the mutation model assumptions on which the estimate is based) have not been considered in detail yet and the problems are often exacerbated in fish species, as some species show very large numbers of alleles at microsatellite loci. These issues and others, e.g. null alleles, are reviewed and possible solutions are proposed     

Microsatellite analysis of kinkajou social organization

Kinkajou social groups generally consist of one adult female, two males, one subadult and one juvenile. Based on analysis of variation in 11 microsatellite loci, we assess the degree of kinship within and between four social groups totaling 25 kinkajous. We use exclusion and likelihood analyses to assign parents for seven of the eight offspring sampled, five with >/= 95% certainty, and two with >/= 80% certainty. Five of six identified sires of group offspring came from the same social group as the mother and pup. Adult males and females within a group were unrelated and subadults and juveniles were offspring of the group adults, suggesting a family structure. All five identified paternities within a social group were by the dominant male of the group. However, this copulation asymmetry does not necessarily reflect cooperation due to kinship ties between the two adult males within a group as one of two adult male pairs sampled was unrelated. Neighbouring male kinkajous were more closely related to each other than neighbouring female kinkajous, suggesting that females disperse more often or farther than males.     

Microsatellite characterization of Cimarron Uruguayo dogs

Various genetic markers, including microsatellites, have been used to analyze the genetic polymorphism and heterozygosity in canine breeds. In this work, we used nine microsatellite markers to investigate the genetic variability in Cimarron Uruguayo dogs, the only officially recognized native canine breed in Uruguay. DNA from 30 Cimarron Uruguayo dogs from northeastern and southern Uruguay was analyzed. The allelic frequencies for each microsatellite, the genetic variability and the consanguinity were calculated, as were the polymorphic information content (PIC) and the probability of exclusion (PE). All of the microsatellites studied were polymorphic. FH 2361, FH 2305 and PEZ 03 were the most informative, with PIC values > 0.7, in agreement with results for other canine breeds. The PE values for the markers were within the ranges previously described and were generally greater for microsatellites with higher PIC values. The heterozygosity value (0.649) was considered high since only nine microsatellites were analyzed. Compared with data for other breeds, the results obtained here indicate that Cimarron Uruguayo dogs have high genetic diversity.