荧光标记的染色体原位杂交及其在人类基因组研究中的…

ＦＩＳＨ技术是８０年代开始发展起来的一种新的定位技术,在人类基因组研究中得到了广泛的应用,通过中期染色体的ＦＩＳＨ可以进行ＳＣＰ,Ｃｏｓｍｉｄ和ＹＡＣ的染色体定位,嵌合克隆的鉴别。通过间期核的ＦＩＳＨ可能在５０ｋｂ的分辨率下进行基因作图;最新的研究进展已可以进行伸展的染色质丝的ＦＩＳＨ,直接测量基因的长度,从而达到高精度基因作图的目的。总之,随着ＦＩＳＨ技术本身的发展,它将在人类基因组研究中发挥更     

BAC-FISH在植物基因组研究中的应用

细菌人工染色体与荧光原位杂交合成技术(BAC-FISH)是90年代开始发展起来的一种新的定位技术.由于该技术较常规荧光原位杂交(FISH)技术的信号检出率高得多,近年来在植物基因组研究中得到了越来越多的应用.运用该技术已将一些重要的功能基因定位到相应植物染色体上.     

FISH技术在贝类分子生物学研究中的应用

在牡蛎和其它的海产贝类中,基因组研究的许多重要领域,如：利用非整倍体在牡蛎种间进行基因转移,三体牡的分离,牡蛎和其它贝类的连锁图的建立,三倍体的基因组稳定性和染色体缺失的分析等在缺少可靠的方法鉴定染色体而受到了限制,传统的带型技术很难鉴定牡的染色体。一种新的生物学技术－荧光原位杂交（FISH）为其提供了新的机遇。通过将DNA序列直接杂交到染色体上,FISH不仅是鉴定染色体的一个有力的工具,也是许多基因组研究如基因定位的一种有效的方法,结合最新研究成果,概述了FISH技术在贝类中的应用背景、现状和展望。     

植物荧光原位杂交技术的发展及其在植物基因组分析中的应用

荧光原位杂交(FISH)是在染色体、间期核和DNA纤维上定位特定DNA序列的一种有效而精确的分子细胞遗传学方法。20年来,植物荧光原位杂交技术发展迅速:以增加检测的靶位数为目的,发展了双色FISH、多色FISH和多探针FISH鸡尾酒技术;为增加很小染色体目标的检测灵敏度,发展了BAC-FISH和酪胺信号放大FISH(TSA-FISH)等技术;以提高相邻杂交信号的空间分辨力为主要目的,发展了高分辨的粗线期染色体FISH、间期核FISH、DNA纤维FISH和超伸展的流式分拣植物染色体FISH技术。在植物基因组分析中,FISH技术发挥了不可替代的重要作用,它可用于:物理定位DNA序列,并为染色体的识别提供有效的标记;对相同DNA序列进行比较物理定位,探讨植物基因组的进化;构建植物基因组的物理图谱;揭示特定染色体区域的DNA分子组织;分析间期核中染色质的组织和细胞周期中染色体的动态变化;鉴定植物转基因。     

外源基因在转基因油菜染色体上的定位及分析

原位杂交技术 ( in situ hybridization,ISH)是基因定位的主要技术之一。近来 ,随着植物细胞染色体制片技术的发展 ,以及酶联放大检测系统的采用 ,在植物中已有低拷贝和单拷贝甚至小于 1 kb的 DNA序列定位的成功报道 [1 ,2 ]。染色体原位杂交技术不仅可以用于基因的物理作图 ,而且可以用来对转基因植物中的外源基因进行染色体定位 [3 5] 。研究表明 ,外源目的基因在转基因植物中的表达与整合位点有关 [6] 。因而 ,进行外源基因在转基因植物染色体上的定位以及研究外源基因的整合位点与表达之间的关系 ,对于开发和利用转基因植物具有重要…     

致病基因的定位候选克隆

基因组研究的迅猛发展,使我们有必要重新审视致病基因克隆的各种策略与技术,以及人类基因组研究在致病基因克隆中的作用。定位候选克隆基因策略强调充分利用已知的细胞遗传学、医学遗传学、分子遗传学、分子生物学和生物化学知识,特别是人类基因组研究的最新成果,综合功能克隆、定位克隆与传统候选基因研究的策略,分离鉴定致病基因。今天的定位克隆已几乎不再需要染色体步移,甚至有可能避开cDNA筛选。     

用人/啮齿类体细胞杂种系及荧光原位杂交将人类新基因ZNF191定位于染色体18q12.1

本文以锌指蛋白ZNF191全长cDNA为探针,与人/啮齿类体细胞杂种系DNA杂交,将这个新的人类锌指基因定位于18号染色体。又用该cDNA筛选人基因组DNA lambda/DASH文库,以获得的DNA片段为探针,进行人染色体荧光原位杂交(FISH)分析,将ZNF191基因精细定位在染色体18q 12.1区带。依据有关遗传连锁分析和等位基因荧光原位杂交(FISH),将ZNF191精确定位于人染色体18q12.1。通过遗传连锁及染色体杂合性丢失分析.目前已知多种遗传病和肿瘤与这个区域相关。因此,ZNF191基因可作为这些疾病或肿瘤的候选相关基因。     

应用荧光原位杂交技术检测人β^E珠蛋白基因小鼠染色体上的整合状态

为将荧光原位杂交技术应用于基因定位研究中,探讨一种能有效地检测转基因动物染色体上外源基因整合状态的实验方法,对小鼠腹腔注射秋水仙素后,取转基因小鼠骨髓制备中期染色体,将传统的FISH方法加以改进,检测外源基因在转基因小鼠染色体上的整合状态。检测结果表明,外源人β^E珠蛋白基因已稳定地整合于小鼠染色体上,FISH能直观地反映外源基因在转基因动物染色体上的整合状态,该方法可对转基因动物及基因转移研究中的外源基因整合反进行染色体定位检测。     

薄片牡蛎的核型及18S-28S核糖体rRNA基因的染色体定位

以薄片牡蛎(Dendostrea folium)成体鳃组织为材料制备有丝分裂中期染色体标本,对其染色体核型进行了分析,并运用荧光原位杂交技术(FISH)将18S-28S核糖体RNA基因定位于中期染色体上。FISH探针是通过PCR扩增介于18S-28S rRNA基因之间的ITS和5.8S rRNA基因序列,并在PCR扩增过程中掺入了Biotin-11-dUTP进行生物素标记。结果显示,薄片牡蛎的单倍染色体数目为n=10,全部为中部着丝粒染色体。与大多数已知巨蛎属牡蛎的染色体核型相似。ITS探针在薄片牡蛎中期分裂体相上产生两簇FISH信号,分别杂交于2号染色体短臂的近端粒区域。本研究首次报道了薄片牡蛎的中期染色体核型以及18S-28S核糖体RNA基因在染色体上的定位。     

运用FISH技术将SSR标志定位于染色体上的研究

目的:运用FISH将SSR标记定位于染色体上,并确定其具体属于哪条染色体.方法:从全基因组测序数据库中挑选SSR标记,再将该序列到Fosmid库中进行序列比对,得到末端序列与SSR序列相同的Fosmid,再利用该Fosmid制作荧光原位杂交的探针,将该探针运用FISH技术杂交到染色体上,同时结合Tpy1,Tpy3序列识别其具体位于哪条染色体上.结果:在荧光显微镜下可以直接观察到探针在染色体上的结合位点,利用相关拍摄软件就可以对其进行拍照.本实验得到了较好的FISH杂交图片,并结合Tpy1,Tpy3成功的将其定于黄瓜五号染色体上.结论:FISH技术可以让人直接观察到探针在染色体上的位置,运用此方法能将可用于进行分子遗传图谱整合的SSR标记准确定位于染色体上.本研究中,在Tpy1,Tpy3探针的帮助下,我们更直接确认了该标记位于黄瓜的第五号染色体上.这使该标记对黄瓜分子辅助育种与黄瓜分子遗传图谱的整合,可以提供直接而有用的帮助.     

Evidence of chromosomal inversion using fluorescence in situ hybridization to stretched DNA

The resolution of fluorescence in situ hybridization techniques (FISH) can be improved using techniques of DNA stretching. The so-called DIRVISH technique has been used to demonstrate the existence of an inversion involving a small chromosomal segment of the long arm of chromosome 14. This inversion was suspected, but not proven, in patients with familial Alzheimer disease. Two-colour FISH using YAC and cosmid probes allowed us to limit the rearranged region around YAC 964e2, which encompasses the Presenilin 1 (PR1) gene. The existence of small-sized inversions within the genome becomes, thus, open to microscope analysis.     

Analysis of pericentromeric chromosome 21 specific YAC clones by FISH: Identification of new markers for molecular-cytogenetic application

Fluorescence in situ hybridization (FISH) of chromosome 21 specific yeast artificial chromosome (YAC) clones after Alu-PCR (polymerase chain reaction) amplification has been used to find new region-specific DNA probes for the heterochromatic region of chromosome 21. Six overlapping YAC clones from a pericentromeric contig map (region 21cen-21q11) were analyzed. Four YAC clones were characterized as hybridizing to several chromosomal locations. They are, therefore, either chimeric or shared by different chromosomes. Two of them containing alphoid satellite DNA, are localized at the centromeric regions of chromosomes 13 and 21 (clone 243A11), and on 13cen, 21cen and 1q3 (clone 781G5); the two others are localized at both 21q11 and 13q2 (clone 759D3), and at 18p (clone 770B3). Two YACs were strongly specific for chromosome 21q11 only (clones 124A7 and 881D2). These YACs were used effectively as probes for identifications of chromosome 21 during metaphase and interphase analysis of 12 individuals, including three families with Down syndrome offspring, and 6 amniocyte samples. The location of YAC clones on 21q11 close to the centromeric region allows the application of these clones as molecular probes for the analysis of marker chromosomes with partial deletions of the long arm as well as for pre- and postnatal diagnosis of trisomy 21 when alphoid or more distal region-specific DNA probes are uninformative. Overlapping YAC clones covering human chromosome 21q may be systematically used to detect a set of band-specific DNA probes for molecular-cytogenetic application.     

Localization of Two Potassium Channel β Subunit Genes, KCNA1B and KCNA2B

The gating properties and current amplitudes of mammalian voltage-activatedShakerpotassium channels are modulated by at least two associated β subunits (Kvβ1.1 and Kvβ1.2). The human Kvβ1.1 gene (KCNA1B) resides on chromosome 3, as indicated by somatic cell hybrid mapping. More precise localization of KCNA1B to 3q26.1 was obtained with fluorescencein situhybridization (FISH) and was corroborated by PCR screening of the CEPH YAC library. The human Kvβ1.2 gene (KCNA2B) resides on chromosome 1, as indicated by somatic cell hybrid mapping, and has been localized by FISH to 1p36.3.     

Region-specific YAC banding and painting probes for comparative genome mapping: implications for the evolution of human chromosome 2

To date, several hundred nonchimeric yeast artificial chromosomes (YACs) from the Centre d'Étude du Polymorphisme Humain containing polymorphic sequence-tagged sites have been mapped by fluoresence in situ hybridization (FISH) on human metaphase chromosomes. Because they carry an average of 1 Mb of human genomic DNA, CEPH YACs generate high-intensity in situ hybridization signals. The available set of cytogenetically and genetically anchored YACs, approximately one every 5–10 cM evenly spaced over almost the entire human genome, provides complex region-specific probes for molecular cytogenetics. YAC probes can be adapted with unlimited flexibility to specific FISH applications such as the study of chromosomal evolution. We have generated representational probes for YAC banding and painting of human chromosome 2 and its great ape homologs. Convergent inversions were found in the pericentric region of the gorilla and orangutan homologs of chromosome 2p.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Characterization of Two Chromosome 12 Cosmid Libraries and Development of STSs from Cosmids Mapped by FISH

We have constructed and characterized two related human chromosome 12-specific cosmid libraries. DNA from flow-sorted chromosomes from a somatic cell hybrid was cloned into a cosmid vector. Approximately 61% of the cosmids in the nearly 26,200 member arrayed libraries (LLt2NC01 and LLt2NC02) contain human DNA inserts, and 31% of the cosmids derived from human DNA contain CA repeats. One hundred and fifty-two cosmids isolated from the libraries have been mapped by fluorescence in situ hybridization (FISH). Cosmids containing human DNA inserts were localized by FISH exclusively to chromosome 12, confirming the chromosomal specificity of the libraries. The cosmids have been localized to all parts of this chromosome, although some regions are more highly represented than others. Partial sequence information was obtained from 44 mapped cosmids, and oligonucleotide primer pairs were synthesized that define unique sequence tagged sites (STSs). These mapped cosmids, and unique STSs derived from them, provide a set of useful clones and primer pairs for screening YAC libraries and developing contigs centered on regions of interest within chromosome 12. In addition, 120 of the mapped cosmids contain CA repeats, and thus they also provide a useful resource for defining highly polymorphic simple tandem repeat elements that serve as genetic markers for linkage analysis and disease gene localization.     

人类YAC库PCR三维筛选体系的建立及质量考核

为了在知道某个区域、位点、基因或ＤＮＡ片段的部分信息后,能从ＣＥＰＨＹＡＣ库中筛选出与其对应的ＹＡＣ克隆,为进一步研究奠定基础,需要建立一个筛选体系。本文概述了这一筛选体系的建立过程。随后,用两对与已知基因对应的引物进行了筛选验证工作,证明了这一体系的可用性,同时提出了以后筛选的途径,即首先筛选ＹＡＣ库的ＭｅｇａＹＡＣ部分,并以５块板为一组进行筛选。另外,运用荧光原位杂交技术（ＦＢＨ）,对ＣＥＰＨＹＡＣ库的质量及其第一代人类基因组物理图谱进行了考察。我们取２６个ＹＡＣ克隆进行ＦＩＳＨ定位,结果其中嵌合体１３个,占５０％。定位错误的克隆有６个,占２３％。非嵌合体且定位正确的共９个,占３５％。     

Physical Mapping of the Human Connexin 40 (GJA5), Flavin-Containing Monooxygenase 5, and Natriuretic Peptide Receptor A Genes on 1q21

The human connexin 40 gene (Cx40; HGMW-approved symbol GJA5), a gap junction protein, was mapped previously to 1pter–q12 by analysis of somatic cell hybrids. In the development of a yeast artificial chromosome (YAC) contig at 1q21, a YAC end clone contained the entire 5′-untranslated region and 42 bases of coding region of the Cx40 gene. Using a sequence-tagged site (STS) developed from this sequence as well as amplimers for the natriuretic peptide receptor A (NPR1) and flavin-containing monooxygenase 5 (FMO5) genes, the order NPR1–FMO5–Cx40 was established, with Cx40 being the most telomeric. Single-color FISH using PAC clones containing the Cx40 and NPR1 STSs localized these genes to 1q21.1.     

An anchored YAC-STS framework for the rat genome

We report here the first YAC-STS framework for the rat genome. A total of 417 anchor microsatellite markers were used to screen a 10-fold redundant YAC library. One or more unambiguous YACs were identified for 372 markers. Assuming the genetic length of the rat genome to be 2,000 cM (Bihoreau et al. 1997b), the YAC-STS framework will provide, on average, one informative YAC clone every 5.4 cM. A total of 111 anchor markers used in this study were derived from known gene regions. We also demonstrated one of the important and immediate uses of this YAC-STS framework, which is to establish a correlation between the genetic and cytogenetic maps in the rat through FISH analysis.     

人类人工染色体作为转基因载体的应用前景

自1997年首次成功构建人类人工染色体(human artificial chromosome,HAC)以来,对其理论、方法学问题的研究一直就是人们关注的焦点,并引起了科学家们的极大兴趣,目前已能采用不同的方法获得多种类型的HAC。与酵母人工染色体（YAC）、细菌人工染色体（BAC）等相比,HAC不整合到细胞的基因组中,以一个独立的功能性染色体单位而存在,并在细胞中进行正常的有丝分裂和减数分裂。迄今的研究表明：HAC可以携带大片段基因组DNA,是研究人类基因表达和调控、染色体功能基本单元的重要工具,也是建立HAC动物模型的重要手段。在未来的基因治疗方面有着广阔的应用前景。