基因差异表达研究方法的探讨--从DD-PCR到cDNA RDA

ＤＤ－ＰＣＲ和ｃＤＮＡ ＲＤＡ都是近年发展起来的新技术,它们被应用于生物及医学领域,在基因差异表达和筛选差异基因的研究中有着广泛的前景。本文论述了ＤＤ－ＰＣＲ及ｃＤＮＡ ＲＤＡ的基本技术策略,并对两者进行了比较分析。     

降低差异显示-PCR假阳性和提高重复性的几种策略

简要介绍了几种提高mRNA差异显示重复性和降低假阳性的策略. mRNA差异显示技术从建立时起就引起了科学家的广泛兴趣, 是克隆不同生理或病理过程中差异表达基因的一种高灵敏度、简单、有效的方法.但mRNA差异显示因其假阳性率高和重复性低,限制了其在生命科学中的应用.     

PCR相关技术在植物病毒检疫检测中的应用

PCR和建立在PCR基础上的分子生物学技术以其灵敏、快速、简便等优点广泛应用于植物病毒的检测。阐述反转录聚合酶链式反应、免疫捕捉反转录PCR、PCR-单链构型多态性、实时荧光定量PCR、差异显示PCR和巢式PCR等相关技术的原理及其在植物病毒检测中的应用现状,以期为我国植物病毒的检疫检测提供有益参考。     

Early differential gene expression of rat lung after exposure to paraquat

Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.     

Comparison of droplet digital PCR and quantitative real-time PCR for examining population dynamics of bacteria in soil

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R ²?=?1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0?~?5?×?10⁸ and 0?~?5?×?10⁷ cells per gram of soil, respectively (n?=?5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract^?1 as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA^?1) at ≥5?×?10⁵ MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y?=?a?·?x) revealed excellent quantitative agreement between the two technologies (a?=?0.98, R ²?=?0.97 in the CupMBT set and a?=?0.90, R ²?=?0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.     

Real-time PCR: what relevance to plant studies?

The appearance of genetically modified organisms on the food market a few years ago, and the demand for more precise and reliable techniques to detect foreign (transgenic or pathogenic) DNA in edible plants, have been the driving force for the introduction of real-time PCR techniques in plant research. This was followed by numerous fundamental research applications aiming to study the expression profiles of endogenous genes and multigene families. Since then, the interest in this technique in the plant scientist community has increased exponentially. This review describes the technical features of quantitative real-time PCR that are especially relevant to plant research, and summarizes its present and future applications.     

From immunoglobulin gene fingerprinting to motif-specific hybridization: advances in the analysis of B lymphoid clonality in rheumatic diseases

In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.     

From immunoglobulin gene fingerprinting to motif-specific hybridization: advances in the analysis of B lymphoid clonality in rheumatic diseases

In rheumatic diseases, autoantibody-producing cells of interest are often hidden in a polyclonal B-lymphocyte population. Immunoglobulin gene fingerprinting is a useful approach to screen for expanding clones and to detect recirculation between different locations. The gene fingerprinting approach and the Southern blot technique have been amalgamated, using electrophoretic transfer of a PCR product from an acrylamide gel onto a nylon membrane followed by hybridization with specific oligonucleotide probes. In contrast to conventional fingerprinting, the authenticity of immunoglobulin genes can be confirmed, individual genes can be detected and handling radionucleotides can be avoided. Also, the membrane may be reused for further investigations.     

A strategy for single site PCR amplification of dsDNA: priming digested cloned or genomic DNA from an anchor-modified restriction site and a short internal sequence

Amplification of dsDNA by polymerase chain reaction (PCR) has been limited to those instances in which segments of known sequence flank the fragment to be amplified. A strategy for the PCR amplification of cloned or genomic dsDNA that necessitates sequence information from only a single short segment (single site PCR) has been devised. The region of known sequence may be located at any position within or adjacent to the segment to be amplified. The basic procedure for amplification consists of 1) digestion of dsDNA with one or more restriction enzymes, 2) ligation with a universal anchor adaptor and 3) PCR amplification using an anchor primer and the primer for the single site of known sequence. The anchor adaptor is designed in such a way as to facilitate the amplification of only those fragments containing the sequence of interest. We have demonstrated the utility of this technique by specifically amplifying and directly sequencing antibody variable region genes from cloned dsDNA and from genomic DNA.     

用差异显示PCR法筛选与血管外膜细胞表型转化相关的基因

为筛选血管外膜成纤维细胞（adventitial fibroblast,AF）与肌成纤维细胞（myofibroblast,MF）间表型转化有关的基因,实验建立了大鼠胸主动脉AF和MF两种细胞模型,用差异显示聚合酶链反应（DD－PCR）技术获得表达差异片段,对差异片段进行克隆和测序分析,并用定量PCR和Northern blot对差别显示结果进行验证。用反义核酸转染技术观察骨桥蛋白（osteopontin,OPN）对AF迁移的影响。结果表明,两种表型细胞存在明显的基因表达差异,其中一个在MF下调的差异片段与GenBank中NADH脱氢酶亚单位5（NADH dehydrogenase subunit 5,Nd5）基因高度同源。另一个在MF上调的差异片段与OPN基因同源。上述差异表达结果被定量PCR及Northern blot证实。此外还有4个表达序列标志（expressed sequence-tag,EST）在GenBank中未查到同源序列。反义OPN寡脱氧核甘酸可抑制AF的迁移活动。结果提示,AF转化为MF可能与ND5基因下调、OPN上调及其它未知基因的表达改变有关。应用反义技术适度抑制OPN表达在防治血管重塑中具有重要作用。     

133 new gene localizations on the rabbit cytogenetic map

Rabbit (Oryctolagus cuniculus), besides its interest for medical research and biotechnological applications, has a small agronomic production in southern European countries. However, it is still a "map-poor" species with about 80 genes mapped. Recently, useful tools for research on this species have been developed, such as heterologous human-rabbit chromosome painting data and a rabbit BAC library. In this study, our aim is to enrich the rabbit cytogenetic map using the FISH technique. Towards this, we have used cDNAs (rabbit and non rabbit) present in the public databases to determine intra-exon primers used to screen our three-genome equivalent BAC library, by standard PCR directly on DNA pools, and by hybridization of high-density filters. 133 BAC clones containing the genes of interest were isolated and FISH-mapped to the rabbit chromosomes. We present the localization of new genes on all rabbit chromosomes except OCU20 and OCUY and some preliminary data on the rabbit/human comparative map. In addition, this set of BAC clones quite regularly distributed on the rabbit genome will be useful to isolate microsatellites, in order to construct a first generation genetic map.     

Advances in <Emphasis Type="Italic">Drosophila</Emphasis> gene targeting and related techniques

Functional biological research has benefited tremendously by analyses of the phenotypes of mutant organisms which can be generated through targeted mutation of genes. In Drosophila, compared with random mutagenesis methods gene targeting has gained its popularity because it can introduce any desired mutation into a gene of interest. However, applications of gene targeting have been limited because the targeting efficiency varies with different genes, and the time and labor of targeting procedure are intensive. Nevertheless, improvement of gene targeting and development of its variant technologies have received much attention of scientists. Here we review recent progress that has been made in expanding the applications of gene targeting, which include the ΦC31 integration system and zinc-finger nucleases induced gene targeting, and new strategies that generate more efficient and reliable gene targeting.