差异显示技术(DD-PCR)及其应用

高等植物在其生命活动过程中,由于基因的选择性表达,决定了高等植物生命活动的多样性和连续性。在植物的生长、发育、衰老和死亡过程中,都有不同的基因起作用。因此,研究基因的选择性表达,掌握其对植物生命活动的调控,克隆新基因,是分子生物学的重要课题.1992年,Liang和Pardee[1]首次提出差异显示技术（DD－PCR）,并且利用这一技术克隆了几个基因。由于该技术具有快速、灵敏、简单和可分析低丰度mRNA的优点[2],很快成为克隆新基因和研究植物基因表达的有力工具c这项技术最初用于医学研究上,近年来已开始在高等植物研究中应用…     

简并PCR技术及其在基因克隆中的应用

本文简要介绍简并PCR技术,包括什么是简并引物,如何设计简并引物,进行简并PCR的反应条件,应用简并PCR获得全长基因的方法和简并PCR技术的应用范围,并对简并PCR技术的局限性及其新进展进行讨论。在此基础上,简述基因的克隆策略以及简并PCR技术在基因克隆中的应用。简并PCR技术是寻找和发现“新”基因或蛋白质家族新成员的一种非常有用的工具。 Abstract:Degenerate PCR is introduced in this paper,including what is degenerate PCR,how to design degenerate primers,how to optimize degenerate PCR parameters,how to applying degenerate PCR to obtain full-length gene and which fields can apply degenerate PCR.The limits and recent advances of degenerate PCR are also discussed.Based on this introduction,strategies of gene cloning and applications of degenerate PCR in gene cloning are summarized in brief.Degenerate PCR is a very useful tool for searching and discovering new genes and new members of a protein family.     

PCR快速筛选重组克隆方法的建立

目的：建立一种PCR技术快速筛选重组克隆的方法。方法：用Triton裂解菌落作为模板,以pMD-18T载体多克隆位点设计的引物与目的基因猪β-INF引物设计不同组合,PCR扩增待检重组克隆。结果：PCR技术快速筛选出猪β-INF重组克隆并鉴定了插入方向。结论：在采用PCR方法直接使用细菌菌落参与反应可以快速筛选重组克隆。     

一株链霉菌菌株NCPC-1020中棘白霉素B脱酰基酶基因的克隆与功能分析

【目的】从一株土壤放线菌来源的野生型链霉菌菌株NCPC-1020中克隆一个具有棘白霉素B脱酰基酶活性的新基因。【方法】采用Degenerate和TAIL PCR两种方法,从链霉菌菌株NCPC-1020基因组中快速克隆获得了该基因序列,然后将基因在变铅青链霉菌TK24中进行异源表达,并进行全细胞催化底物脱酰基反应,采用LC-MS检测反应产物。【结果】LC-MS检测证实,棘白霉素B结构中脂肪链被酶促水解,从而证实该基因具有脱酰基酶活性。【结论】采用Degenerate以及TAIL PCR的方法能够快速获得未知功能的新基因。此基因的克隆,奠定了进行半合成棘白霉素类药物的研发基础。     

简并PCR及其应用

简并PCR技术是根据同源蛋白的氨基酸序列扩增到同源基因的核苷酸序列,具有独特的优点。成功进行简并PCR的关键是设计好两组引物库和对反应条件进行优化。由于简并PCR自身的特点,在新基因的克隆、基因表达检测、病毒检测和基因组研究中有其广泛而独特的应用。     

运用多种策略改良差异显示PCR

目的：改进差异显示PCR技术,提高其在筛选差异表达基因方面的效率。方法：①采用单碱基金铆钉引物;②增加引物长度;③提高PCR反应的严谨性;④应用同步重复测序电泳。结果：减少经典方法的工作量,降低了非特异性和假阳性率。用改良技术研究热适应大鼠下丘脑基因的差异表达,发现了一个差异表达基因片段,斑点杂交证实为阳性片段。结论：改进后的差异显示PCR技术是一种研究基因差异表达从而发现新基因或基因新功能的有效方法。     

用差异显示PCR技术克隆冬小麦春化设定cDNA克隆实验系统的建立

差异显示PCR技术是一种新近发展起来的在真核细胞中检测与克隆特异表达基因的手段。本文建立了用此技术克隆春化相关基因的研究系统,并提供了诸如总RNA的提取、污染DNA的去除、sscDNA的逆转录、PCR参数、扩增cDNA的电泳、特异cDNA的收集与重扩增等在方法上的细节。用这种技术分离了一个仅在春化20天这一特异时期表达的春化设定cDNA克隆VPC28。这些结果也适用于更加广泛的类似研究。     

肥胖基因的分离及其在大肠杆菌中的表达

利用PCR技术自外周血白细胞染色体DNA中扩增获取了肥胖基因(ob基因)的外显子2和3序列.经过拼接,获得了全长的ob基因编码序列. 测序结果表明,获得的序列与文献报道完全一致.利用PCR技术扩增出成熟蛋白的编码序列,克隆至表达载体pBV220中获得了表达菌株,并对表达产物进行了初步纯化,为进一步研究ob基因产物的功能与应用奠定了基础.     

应用反向PCR克隆慢病毒介导的转基因小鼠整合位点序列

目的：为分析慢病毒介导的转基因小鼠中外源基因整合位点的信息,应用反向PCR克隆整合位点序列。方法：小鼠基因组总DNA酶解和自连接后,针对慢病毒载体的特点在LTR附近设计一组特异的PCR引物,优化半巢式PCR的各种参数,提高整合位点序列克隆的效率。结果：克隆了分别携带绿色荧光蛋白（GFP）和转铁蛋白（TF）基因的慢病毒介导的转基因小鼠家系7只小鼠中10个外源基因整合位点序列。结论：本方法可用于慢病毒介导的转基因小鼠整合位点序列的克隆,为分析整合位点与外源基因表达之间的关系等提供了科学依据。     

用PCR法直接快速筛查重组阳性克隆

本研究目的是应用PCR法快速筛查插入有苯丙氨酸脱氨酶（PAL）cDNA重组阳性克隆,用于PCR扩增的引物是位于载体pET23b启动子处的T7启动子引物和位于目的基因PALc DNA3’端终止密码TAA处的引物,以灭菌吸头挑一单菌落加PCR体系扩增。结果：在筛查的3个克隆中,有2个阳性克隆,并且插入方向正确,经DNA序列测定得到进一步证实,结果表明,以PCR方法筛查重组阳性克隆,可以简便快速鉴定插入片段的大小和方向,不需提取质粒。     

Polymerase chain reaction and its applications: special emphasis on its role in embryo sexing

The polymerase chain reaction (PCR) has developed into one of the most promising methods for in vitro enzymatic amplification of DNA and has found widespread application in DNA cloning, sequencing and mutagenesis related studies. This innovative technique can selectively amplify a single target DNA molecule a billion-fold in a span of a few hours. Amplification of specific DNA sequences by PCR is useful in identification of sex, novel genes, pathogens and diseases. PCR has facilitated the establishment of evolutionary relationships among species and in revealing structural intricacies of single cells. In this article we review some of the major advances and applications of PCR, especially, its role in embryo sexing.     

Cloning and mapping of telomere-associated sequences from Hordeum vulgare L.

Summary We present a novel approach to the efficient cloning of telomere-associated sequences and demonstrate its application to the cloning and mapping of these sequences from barley. The method is a modification of the Vectorette PCR technique and allows specific amplification and cloning of subtelomeric sequences. Telomere-associated sequences isolated from barley include hypervariable, repetitive sequences. Polymorphisms detected by subtelomeric markers behaved as Mendelian factors and were mapped to the most distal positions of barley chromosomes 1, 2, and 4.     

Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition

Serial analysis of ribosomal sequence tags (SARST) is a novel technique for characterizing microbial community composition. The SARST method captures sequence information from concatemers of short 16S rDNA polymerase chain reaction (PCR) amplicons from complex populations of DNA. Here, we describe a similar method, serial analysis of V6 ribosomal sequence tags (SARST-V6), which targets the V6 hypervariable region of bacterial 16S rRNA genes. The SARST-V6 technique exploits internal primer sequences to generate compatible restriction digest overhangs, thereby improving upon the efficiency of SARST. Serial analysis of V6 ribosomal sequence tags of bacterial community composition in hydrothermal marine sediments from Guaymas Basin resembled results of cloning and sequencing of single, full-length PCR products from ribosomal RNA genes of the same microbial community. Both methods identified the same major bacterial groups, but only SARST-V6 recovered thermodesulfobacteria and gamma-proteobacteria sequences, while only full-length PCR product cloning recovered candidate division OP11 se-quences. There were differences in the relative frequencies of some phylotypes. The disparities reflect differences in the amplicon pool obtained during initial amplification that may result from different primer affinities or DNA degradation. These results demonstrate the utility of SARST-V6 in collecting taxonomically informative data for high-throughput analysis of microbial communities.     

Targeted gene walking polymerase chain reaction.

We describe a modification of a polymerase chain reaction method called 'targeted gene walking' that can be used for the amplification of unknown DNA sequences adjacent to a short stretch of known sequence by using the combination of a single, targeted sequence specific PCR primer with a second, nonspecific 'walking' primer. This technique can replace conventional cloning and screening methods with a single step PCR protocol to greatly expedite the isolation of sequences either upstream or downstream from a known sequence. A number of potential applications are discussed, including its utility as an alternative to cloning and screening for new genes or cDNAs, as a method for searching for polymorphic sites, restriction endonuclease or regulatory regions, and its adaptation to rapidly sequence DNA of lengthy unknown regions that are contiguous to known genes.     

Mark–recapture cloning: a straightforward and cost‐effective cloning method for population genetics of single‐copy nuclear DNA sequences in diploids

We describe a simple protocol to reduce the number of cloning reactions of nuclear DNA sequences in population genetic studies of diploid organisms. Cloning is a necessary step to obtain correct haplotypes in such organisms, and, while traditional methods are efficient at cloning together many genes of a single individual, population geneticists rather need to clone the same locus in many individuals. Our method consists of marking individual sequences during the polymerase chain reaction (PCR) using 5′‐tailed primers with small polynucleotide tags. PCR products are mixed together before the cloning reaction and clones are sequenced with universal plasmid primers. The individual from which a sequence comes from is identified by the tag sequences upstream of each initial primer. We called our protocol mark–recapture (MR) cloning. We present results from 57 experiments of MR cloning conducted in four distinct laboratories using nuclear loci of various lengths in different invertebrate species. Rate of capture (proportion of individuals for which one or more sequences were retrieved) and multiple capture (proportion of individuals for which two or more sequences were retrieved) empirically obtained are described. We estimated that MR cloning allowed reducing costs by up to 70% when compared to conventional individual‐based cloning. However, we recommend to adjust the mark:recapture ratio in order to obtain multiple sequences from the same individual and circumvent inherent technical artefacts of PCR, cloning and sequencing. We argue that MR cloning is a valid and reliable high‐throughput method, providing the number of sequences exceeds the number of individuals initially amplified.     

Uncultured soil bacteria are a reservoir of new antibiotic resistance genes

Antibiotic resistance genes are typically isolated by cloning from cultured bacteria or by polymerase chain reaction (PCR) amplification from environmental samples. These methods do not access the potential reservoir of undiscovered antibiotic resistance genes harboured by soil bacteria because most soil bacteria are not cultured readily, and PCR detection of antibiotic resistance genes depends on primers that are based on known genes. To explore this reservoir, we isolated DNA directly from soil samples, cloned the DNA and selected for clones that expressed antibiotic resistance in Escherichia coli. We constructed four libraries that collectively contain 4.1 gigabases of cloned soil DNA. From these and two previously reported libraries, we identified nine clones expressing resistance to aminoglycoside antibiotics and one expressing tetracycline resistance. Based on the predicted amino acid sequences of the resistance genes, the resistance mechanisms include efflux of tetracycline and inactivation of aminoglycoside antibiotics by phosphorylation and acetylation. With one exception, all the sequences are considerably different from previously reported sequences. The results indicate that soil bacteria are a reservoir of antibiotic resistance genes with greater genetic diversity than previously accounted for, and that the diversity can be surveyed by a culture-independent method.     

Cataloging altered gene expression in young and senescent cells using enhanced differential display.

Recently, a novel PCR-based technique, differential display (DD), has facilitated the study of differentially expressed genes at the mRNA level. We report here an improved version of DD, which we call Enhanced Differential Display (EDD). We have modified the technique to enhance reproducibility and to facilitate sequencing and cloning. Using EDD, we have generated and verified a catalog of genes that are differentially expressed between young and senescent human diploid fibroblasts (HDF). From 168 genetags that were identified initially, 84 could be sequenced directly from PCR amplified bands. These sequences represent 27 known genes and 37 novel genes. By Northern blot analysis we have confirmed the differential expression of a total of 23 genes (12 known, 11 novel), while 19 (seven known, 12 novel) did not show differential expression. Several of the known genes were previously observed by others to be differentially expressed between young and senescent fibroblasts, thereby validating the technique.     

细胞内POCR法克隆抗体可变区基因

报道一种克隆免疫脾细胞中抗体可变区基因的新方法。抗原免疫小鼠的脾细胞经分散、固定、渗透处理,直接在细胞内进行反转录,再采用半巢式PCR扩增,获得的序列经测序证明是抗体可变区编码序列。这种不经mRNA纯化、从细胞内直接获取目的的方法既可用于抗体库的建立,亦可用于新基因的快速克隆。     

Molecular analyses of methyl-coenzyme M reductase alpha-subunit (mcrA) genes in rice field soil and enrichment cultures reveal the methanogenic phenotype of a novel archaeal lineage

The diversity of methanogen-specific methyl-coenzyme M reductase alpha-subunit (mcrA/mrtA) genes in Italian rice field soil was analysed using a combination of molecular techniques and enrichment cultures. From 75 mcrA/mrtA clones retrieved from rice field soil, 52 were related to members of the Methanosarcinaceae, Methanosaetaceae and Methanobacteriaceae. However, 19 and four clones formed two novel clusters of deeply branching mcrA sequences, respectively, which could not be affiliated to known methanogens. A new methanogen-specific fingerprinting assay based on terminal restriction fragment length polymorphism (T-RFLP) analysis of fluorescently labelled polymerase chain reaction (PCR) products allowed us to distinguish all environmental mcrA/mrtA sequences via group-specific Sau96I restriction sites. Even genes for the isoenzyme methyl-coenzyme M reductase two (mrtA) of Methanobacteriaceae present in rice field soil were represented by a unique 470 bp terminal restriction fragment (T-RF). Both cloning and T-RFLP analysis indicated a significant representation of novel environmental mcrA sequences in rice field soil (238 bp T-RF). To identify these mcrA sequences, methanogenic enrichment cultures with rice field soil as inoculum were established with H2/CO2 as substrates at a temperature of 50 degrees C, and these were monitored using molecular tools. In subsequent transfers of these enrichment cultures, cloning and T-RFLP analysis detected predominantly SSU rRNA genes of rice cluster I (RC-I), an uncultivated euryarchaeotal lineage discovered previously in anoxic rice field soil. In parallel, both mcrA cloning and T-RFLP analyses of the enrichment culture identified the more frequent cluster of novel environmental mcrA sequences as belonging to members of RC-I. Thus, we could demonstrate the genotype and phenotype of RC-I Archaea by the presence of a catabolic gene in a methanogenic enrichment culture before the isolation of pure cultures.