介绍一种非放射性标记的原位杂交新方法

在病理诊断和科研工作中,原位杂交是一种常用的检测技术。原位杂交组织化学方法是将核酸分子杂交技术与组织细胞原位显示某种特定ＤＮＡｍＲＮＡ及其产物的定性定量及变化规律相结合的一种新技术。目前,在检测特异ｍＲＮＡ的原位杂交组织细胞化学中多采用同位素标记的探...     

荧光原位杂交技术及其在医学诊断上的应用

荧光原位杂交技术是近年来生物学领域发展起来的将经典的细胞遗传学与分子遗传学结合起来一项新技术。该技术具有广泛的应用潜力,在细胞生物学、分子生物学、医学等众多领域快速发展。本文介绍了荧光原位杂交技术的基本原理和操作方法,并对该技术目前的发展状况以其在医学诊断上的应用进行了阐述。     

荧光原位杂交技术的研究进展

荧光原位杂交(FISH)是在染色体、间期细胞核和DNA纤维上进行DNA序列定位的一种有效手段。近年来,围绕提高检测的分辨率和灵敏性,不断将免疫染色、量子点和微流控芯片等物理化学技术引入到荧光原位杂交中,促进了它的快速发展。本文主要综述了荧光原位杂交的基本原理和发展历程,重点介绍了免疫染色-荧光原位杂交(immuno-FISH)、量子点-荧光原位杂交(QD-FISH)和微流控芯片-荧光原位杂交(FISH on microchip)等多种新技术及其检测特点,如快速、灵敏、动态、多样化等。随着荧光原位杂交技术的不断完善与发展,将在细胞遗传学、表观遗传学及分子生物学等领域发挥更加重要的作用。     

论分子生物学技术在环境微生物研究中的应用

随着分子生物学技术在新时期的高速发展,越来越多的新方法、新技术被应用于环境微生物研究,本文简要介绍了荧光原位杂交、变性梯度凝胶电泳、末端限制性酶切片段长度多态性分析、基因芯片技术等在环境微生物研究中的应用,不仅可以在环境微生物研究中最大限度的获取微生物遗传信息,并可以对环境中微生物的多样性进行全面分析。     

基因组原位杂交技术及其在园艺植物基因组研究中的应用

基因组原位杂交(GISH)技术可以鉴定植物多倍体物种起源、杂种亲本染色体来源和组成,分析栽培种与其近缘野生种的亲缘关系,研究减数分裂染色体行为等。基因组原位杂交包括多色基因组原位杂交、比较基因组原位杂交和自身基因组原位杂交等。基因组原位杂交技术的关键步骤是染色体制片、探针制备及长度优化、探针与封阻的浓度比例和杂交后洗脱强度。该文对近年来国内外有关基因组原位杂交技术的发展及其在园艺植物基因组研究中的应用现状进行了综述,并指出随着多种园艺植物全基因组的测定,未来应从基因组信息中寻找更多的染色体特异性标记,结合荧光显带及荧光原位杂交技术,为深入研究园艺植物的起源以及遗传关系鉴定等提供技术支持。     

荧光原位杂交技术及其在植物研究中的应用

荧光原位杂交(FISH)是20世纪生物学领域的一项新技术。FISH应用细胞遗传学和分子生物学的基本原理,作为架设细胞遗传学与分子生物学之间的桥梁,现已被广泛应用于植物学各方面的研究。本文就FISH的基本原理、技术发展及其在植物遗传育种、起源进化、染色体物理图谱构建方面的应用及发展趋势进行了综述。     

荧光原位杂交技术及其在环境微生物生态学中的应用研究

荧光原位杂交技术是一种能够同时对微生物进行定性、定量和研究微生物群落空间分布情况的有力工具。简要介绍了荧光原位杂交技术的方法,并对其在人为创制环境和自然环境中特征性微生物种群及群落生态学中的应用研究进行了讨论,指出了该种技术在应用中存在的问题与缺陷,最后对荧光原位杂交技术在堆肥微生物生态中的应用及与其他方法的组合应用进行了展望。     

原位杂交技术在植物遗传育种上的应用

本文在简要介绍原位杂交技术的基础之上 ,重点介绍了该技术在植物遗传育种领域 ,即在 (1 )异源染色质及染色体畸变检测 ;(2 )植物基因工程及基因表达研究 ;(3)构建植物基因物理图谱 ;(4)染色体RNA研究等方面的应用现状 ,并对原位杂交技术在提高检出率 ,与染色体显带技术结合 ,PCR 原位杂交等方面提了一些见解。     

原位杂交技术在植物遗传育种上的应用

本文在简要介绍原位杂交技术的基础之上,重点介绍了该技术在植物遗传育种领域,即在(1)异源染色质及染色体畸变检测;(2)植物基因工程及基因表达研究;(3)构建植物基因物理图谱;(4)染色体RNA研究等方面的应用现状,并对原位杂交技术在提高检出率,与染色体显带技术结合,PCR-原位杂交等方面提了一些见解。     

在植物粗线期染色体和DNA纤维上的荧光原位杂交技术

介绍了两种荧光原位杂交技术的详细实验步骤。第一种技术是在减数分裂粗线期染色体上的荧光原位杂交,包括从花粉母细胞中制备粗线期染色体和在这种染色体上定位ＤＮＡ序列,其分辨率水平能够达到１００ｋｂ。第二种技术是从植物细胞核中制备ＤＮＡ纤维,并在上面进行原位杂交,能够直接分析ＤＮＡ序列的分子排列关系,其分辨率水平能达到几个ｋｂ。为了说明这两种原位杂交技术在研究基因组和染色体结构、构建高分辨率的ＤＮＡ物理图谱上的能力,将展示用该技术直接分析番茄染色体端粒重复序列和端粒联接重复序列的染色体定位和ＤＮＡ分子排列。     

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.     

GEISHA: an in situ hybridization gene expression resource for the chicken embryo

An important and ongoing focus of biomedical and agricultural avian research is to understand gene function, which for a significant fraction of genes remains unknown. A first step is to determine when and where genes are expressed during development and in the adult. Whole mount in situ hybridization gives precise spatial and temporal resolution of gene expression throughout an embryo, and a comprehensive analysis and centralized repository of in situ hybridization information would provide a valuable research tool. The GEISHA project (gallus expression in situ hybridization analysis) was initiated to explore the utility of using high-throughput in situ hybridization as a means for gene discovery and annotation in chicken embryos, and to provide a unified repository for in situ hybridization information. This report describes the design and implementation of a new GEISHA database and user interface (www.geisha.arizona.edu), and illustrates its utility for researchers in the biomedical and poultry science communities. Results obtained from a high throughput screen of microRNA expression in chicken embryos are also presented.     

Flow cytometric measurement of telomere length

The regulation of telomere length may be involved in the cellular physiology of senescence, reproduction, cancer, immune response to infection, and possibly immune deficiency. The measurement of telomere length, critical to research in this area, has traditionally been performed by Southern blot analysis, which is cumbersome and time consuming. Several alternative methods have been described in recent years. Some, such as pulsed-field electrophoresis, slot blots, and centromere-to-telomere ratio measurements are essentially improvements to the Southern blot technique. However, other methods such as fluorescent in situ hybridization on metaphase chromosome spreads and flow cytometry-based fluorescent in situ hybridization represent a completely new technical approach to the problem. In this review, we compare methods, with particular emphasis placed on flow cytometric techniques for measuring telomere length in situ and identifying potential areas where improvements may still be made.     

斑马鱼整胚原位杂交和显微注射转基因技术的建立(简报)

随着人类基因组框架图的完成,解读大量基因的功能和表达调控成为亟待解决的问题。因此通过模式生物从个体水平、细胞水平和分子水平研究基因功能及其表达调控日益成为一个重要的研究领域。斑马鱼(Brachydanio rerio)是近年来崛起的一种模式生物。它的显著优势在于:个体小,便于大批量饲养;产卵量大,胚胎透明,易于观察和操作;体外发育,发育迅速,2—3天即可完成主要器官的建成;可以方便的进行大规模的诱变和突变型筛选等研究。它已成为脊椎动物胚胎发育遗传学和基因功能研究中理想的模式生物。整胚原位杂交技术是     

Whole amount in situ hybridization and transgene via microinjection in zebrafish]

Zebrafish is new model organism to study development of the vertebrate. In this report, we chose a fragment of gene GATA-1 which specifically expressed in zebrafish hematopoiesis system as probe to carry on whole amount in situ hybridization. Then we transported reporter gene driven by GATA-1 promoter into the embryos via microinjection approach and observed the spatial and temporal expression pattern of GFP. Our results demonstrated that efficient and reliable technology of whole amount in situ hybridization and microinjection in zebrafish was established.     

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.     

The mitotic chromosomes of Notophthalmus (=Triturus) viridescens: Localization of C banding regions and DNA sequences complementary to 18S, 28S and 5S ribosomal RNA

The metaphase chromosomes of Notophthalmus (Triturus) viridescens have been studied by C-banding and in situ hybridization. The chromosomes show the pericentric C-banding seen in many organisms and in addition have interstitial C-bands located a short distance from the pericentric C-bands on each chromosome arm. A few C-bands are seen in telomeric regions. Regions which hybridize in situ with 18S and 28S ribosomal RNA were found on three chromosome pairs. The animals studied fell into three groups with respect to which of the six possible sites showed detectable hybridization with 18S and 28S RNA. Individual animals differed not only in the pattern of in situ hybridization of ribosomal RNA but also in the number of ribosomal RNA cistrons in the genome as measured by saturation hybridization on purified DNA. In situ hybridization showed five pairs of chromosomes which contained DNA complementary to 5S RNA. The four pairs of subtelocentric chromosomes in the N. viridescens karyotype all have 5S DNA in the pericentric regions. The fifth cluster of 5S DNA is in the middle of one arm of the chromosomes in one of the two smallest submetacentric pairs in the genome. The five sites of 5S DNA differ markedly in the level of in situ hybridization with 5S cRNA.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.