应用于染色体步移的PCR扩增技术的研究进展

各种建立在PCR基础上染色体步移的方法能够根据已知的基因序列得到侧翼的基因序列。染色体步移技术主要应用于克隆启动子、步查获得新物种中基因的非保守区域、鉴定T-DNA或转座子的插入位点、染色体测序工作中的空隙填补,从而获得完整的基因组序列等方面。其方法主要有3种：反向PCR的方法,连接法介导的PCR的方法以及特异引物PCR的方法。文章就各种方法进行举例说明并加以分析比较。     

染色体步移技术研究进展

染色体步移技术是一种常用的克隆已知片段旁侧序列的技术.综述了近年来染色体步移技术的发展情况,介绍了结合基因组文库的染色体步移技术和基于PCR的染色体步移技术.同时总结了物理剪切法和限制性内切酶法构建亚克隆文库的优化步骤,以及连接成环PCR法、外源接头介导PCR法和半随机引物PCR法的原理,并且比较分析了他们之间的优缺点,以期对实际操作起到借鉴作用.     

利用染色体步移PCR检测辐射松的单核苷酸多态性

用染色体步移技术(chromosome walking)的基本原理以辐射松(Pinus radiata)肌动蛋白基因(actin)为例,利用获得的EST序列设计定向引物,向上游和下游进行了染色体序列的步移.获得了包括启动子、5′端非编码区和编码区及3′端非编码区辐射松肌动蛋白基因基因组序列2154 bp.通过对200株不同辐射松个体进行PCR扩增及测序,共获得了21个SNPs,其中启动子区域3个,编码区15个,3′端非编码区4个.实验结果为今后染色体步移技术在基因非编码区SNP的检测提供了理论与技术参考.     

线粒体基因组测序策略和方法

本文综述了线粒体基因组测序策略和方法,在传统测序方法中介绍了基于物理分离线粒体DNA的克隆文库测序方法和基于PCR扩增产物的直接测序方法,后者重点介绍了基于长PCR扩增产物的引物步移法和基于总DNA的引物步移法;应用新一代高通量测序方法有基于总DNA样品的方法,包括需要预扩增mtDNA的多物种平行高通量和无需预扩增mtDNA的高通量方法,基于总RNA样品的转录组测序方法等。在实际工作中,选择哪种方法取决于研究规模、样品大小和保存状态、经费情况等。总的来说,基于长PCR扩增产物的引物步移法尤其适合小规模昆虫线粒体基因组研究,而对于大规模线粒体基因组研究来说,NGS技术无疑是省时省力的最佳选择。     

特异性检测胰腺组织表达Cre重组酶转基因小鼠的方法

利用组织特异性分子标志物启动子调控Cre重组酶,研制了6种在不同组织中特异性表达Cre重组酶的转基因小鼠.这些转基因小鼠的基因型鉴定均使用设计在Cre基因编码区的通用引物.为了特异性检测胰腺组织表达Cre重组酶的转基因小鼠,在大鼠胰岛素RIP启动子上和Cre基因上设计1对引物进行PCR扩增,并通过凝胶电泳进行分析.PCR结果显示,设计在Cre基因上的通用引物可以从6种不同组织特异性Cre重组酶转基因小鼠基因组DNA中扩增获得480 bp产物;利用本研究设计的特异性引物可以从胰腺组织表达Cre重组酶转基因小鼠基因组DNA中扩增200 bp的目的条带.这一结果表明,利用特异性引物进行PCR反应,可有效地将胰腺组织表达Cre重组酶转基因小鼠与其他多种组织的Cm重组酶转基因小鼠鉴别开来.     

基于PCR的染色体步移中单引物扩增的克服与非特异引物的利用

基于PCR的染色体步移(PCR-Walking)方法已有许多种,包括反向PCR、连接介导的PCR、随机引物PCR等.在众多的方法中,经常存在由通用引物引起的单引物非特异扩增现象.本文综述了连接介导的PCR-Walking中单引物扩增的形成原理及克服方法.克服单引物扩增主要是使接头引物在DNA两端的接头上只有1个结合位点,从而避开单引物扩增.常用的方法有3′端加氨基修饰的不对称接头、泡泡状接头或Y字型接头及单寡核苷酸接头等方法.还介绍了2种利用通用引物非特异扩增克隆目的序列的方法:引物错配法及基于RAPD原理的单引物PCR法.     

猪β2-AR基因重组质粒的快速筛选

为建立一种高效快速鉴定重组质粒的实验方法,将猪β2-肾上腺素能受体（β2-AR）基因与pET-32C重组,构建β2-AR基因表达载体pET32CAR,以T7启动子引物和猪β2-肾上腺素能受体基因特异性引物为PCR引物,挑取重组质粒转化单菌落直接进行PCR,产物经电泳发现,5个候选克隆中有3个克隆扩增出了一条约2kb的特异性条带,并且插入方向正确,随机选取其中1个克隆用限制性酶切鉴定以及DNA测序验证PCR筛选的正确性,研究结果表明;在采用PCR方法对重组克隆进行鉴定时,可以直接使用细菌菌落参与反应,而无须提取克隆的DNA,与传统的实验方法相比,筛选的时间缩短至3-4h,大大提高了重组DNA的筛选效率。     

利用PCR鉴定四川雉鹑性别

四川雉鹑是我国特有珍稀濒危雉类,为国家Ⅰ级保护动物.本文通过设计引物,扩增四川雉鹑W染色体上雌性特异基因HINTW,利用PCR产物有无对其进行性别鉴定,发现在四川雉鹑中雄性个体无阳性产物,而雌性扩增出500 bp和300 bp 2条条带.应用通用引物扩增四川雉鹑基因组CHD基因,其中雄性个体扩增出约500 bp的条带,而雌性个体则扩增出500 bp和750 bp 2条条带,通过其Z/W染色体上基因片段长度差异可进行性别鉴定.上述两种方法对9个四川雉鹑的性别鉴定结果均一致.     

利用PCR技术构建体外高效转录系统

设计并合成了一对 PCR 反应引物,其5′端引物除含有目的基因5′端序列外,还外加 T7 RNA 聚合酶启动子的17个核苷酸.3′端引物则按常规设计.以染色体 DNA为模板,通过 PCR,可扩增出带有 T7 RNA 聚合酶启动子的目的基因 DNA 片段.以此 PCR 产物为模板,在体外成功实现了高效转录.这是一种快速、简便构建体外高效转录系统的好方法.     

PCR-mtDNA技术鉴别检测不同动物肌肉组织和饲料中鸭源性成分

以鸭肌肉组织DNA为模板,利用PCR-mtDNA技术成功克隆出了鸭mtDNA COIII基因(GenBank Accession No.DQ655706).对所克隆的序列分析表明.其序列包括鸭细胞色素C氧化酶III(COIII)基因全序列784 bp,通过同源性分析可知,动物的线粒体DNA COIII基因是相对保守的,利用此特性设计PCR-mtDNA方法鉴别检测鸭源性成分的特异性引物;以各种动物肌肉组织及饲料DNA为模板进行PCR扩增、经反复验证筛选出只能扩增出鸭DNA的目的片段,而不能扩增出其他动物DNA片段的特异性强、稳定性好的引物P3、P4;利用此引物PCR扩增鸭DNA的特异性片段为226 bp,对PCR产物进行测序分析可知与已克隆的鸭mtDNA COIII基因同源性达到100%,证明了所筛选引物的准确性.通过对不同含量的DNA模板溶液进行PCR扩增的方法,对筛选出的特异性引物P3、P4进行灵敏度试验,结果分析表明灵敏度约为0.001%,证明该PCR方法具有特异性强、灵敏度高的特点,完全可作为鉴别不同动物肌肉组织和饲料中鸭源性成分的方法.     

苹果属小金海棠的遗传多样性初步研究

用RAPD技术建立了苹果属小金海棠（Malus xiaojinensis）2个自然分布区的3个群体内随机选取的30株树（10株／每群体）及其相应的子代实生苗共6个群体、60个样本植株的分子标记。通过对15个3承机引物产生的81条RAPD这的统计,计算了不同群体RAPD多态性带的数目。用TREECON软件分析了不同群体及所有个体间的遗传关系,并用AMOVA技术分析了物种的遗传变异。结果是15个引物在全部分析个体中产生了58条多态性带,平均每引物3.8条。现有分布3个群体及其相应的子代实生苗群体的平均多态性带的数目都为1.5条左右。其中平均多态性带的数目最低的群体仅有0.7条,最高的群体也只有2.5条。遗传关系分析表明,2个自然分布区的不同群体间存在遗传分化现象。AMOVA分析显示小金海棠的遗传变异有相当一部分来源于群体间。     

RAPD早期鉴定平邑甜茶与B9的杂交群体

应用RAPD技术寻找平邑甜茶与B9的差异引物,用于早期筛选以平邑甜茶为母本B9为父本的杂交群体。在用于筛选的72个引物中找到符合要求的4个随机引物共7个特异位点,用这4个随机引物筛选杂交群体的25株个体,鉴定出5个杂交个体：307、308、319、322、324。     

A polymerase chain reaction mediated by a single primer: cloning of genomic sequences adjacent to a serotonin receptor protein coding region.

Under appropriate conditions, specific double-stranded DNA product was generated after amplification of genomic DNA sequences in a polymerase chain-like reaction that contained only a single primer. This type of amplification reaction was performed with a variety of primers and substrate DNAs. In addition to nonspecific heterogeneous products, 5 of 11 primers reproducibly directed synthesis of double-stranded DNA that corresponded to the region of the template that contained the authentic primer annealing site. Three of these amplified products were cloned and their ends were sequenced. All three contained a copy of the primer at both 5' ends, and the position of one of the primers represented the authentic primer binding site. In each case, the location of the second copy of the primer indicated that it had initially hybridized to a partially homologous sequence in the template DNA. This single primer reaction makes it possible to amplify and clone a DNA region of unknown sequence that is adjacent to a known DNA sequence. One of the single primer reaction products described here included sequence to the 5' side of the coding region of a serotonin receptor gene that contained a functional promoter.     

Genetic homogeneity of guava plants derived from somatic embryogenesis using SSR and ISSR markers

To evaluate genetic homogeneity of 1-year-old guava (Psidium guajava L.) plants developed from in vitro somatic embryogenesis, DNA from leaf tissues of seven randomly selected plants along with the mother plant, was isolated and subjected to molecular analysis. A total of six Simple Sequence Repeat (SSR) primer pairs, producing reproducible and clear bands ranging from 100 to 300?bp in size, resulted in amplification of single band (allele), corresponding homozygous individuals. Moreover, of 10 different inter-simple sequence repeat (ISSR) primers screened, six produced resolvable, reproducible and scorable bands. All these ISSRs produced a total of 25 bands, ranging between 300 and 1,200?bp length, and the number of scorable bands, for each primer varied from three to six with an average of 4.1 bands per primer. The amplification products were monomorphic across all the micropropagated plants produced by all SSR and ISSR primers applied. The monomorphic banding pattern in micropropagated plants and the mother plant confirms the genetic homogeneity of the in vitro raised plants and demonstrates the reliability of our in vitro propagation system for guava.     

A New PCR Method: One Primer Amplification of PCR-CTPP Products

Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) is a convenient method for genotyping single nucleotide polymorphisms, saving time, and costs. It uses four primers for PCR; F1 and R1 for one allele, and F2 and R2 for the other allele, by which three different sizes of DNA are amplified; between F1 and R1, between F2 and R2, and between F1 and R2. To date, we have applied PCR-CTPP successfully for genotyping more than 60 polymorphisms. However, it is not rare that PCR does not produce balanced amplification of allele specific bands. Accordingly, the method was modified by attaching a common sequence at the 5' end of two-pair primers and adding another primer with the common sequence in PCR, in total five different primers in a tube for PCR. The modification allowed one primer amplification for the products of initial PCR with confronting two-pair primers, named as one primer amplification of PCR-CTPP products (OPA-CTPP). This article demonstrates an example for an A/G polymorphism of paraoxonase 1 (PON1) Gln192Arg (rs662). PCR-CTPP failed clear genotyping for the polymorphism, while OPA-CTPP successfully produced PCR products corresponding to the allele. The present example indicated that the OPA-CTPP would be useful in the case that PCR-CTPP failed to produce balanced PCR products specific to each allele.     

IRAP and REMAP for retrotransposon-based genotyping and fingerprinting

Retrotransposons can be used as markers because their integration creates new joints between genomic DNA and their conserved ends. To detect polymorphisms for retrotransposon insertion, marker systems generally rely on PCR amplification between these ends and some component of flanking genomic DNA. We have developed two methods, retrotransposon-microsatellite amplified polymorphism (REMAP) analysis and inter-retrotransposon amplified polymorphism (IRAP) analysis, that require neither restriction enzyme digestion nor ligation to generate the marker bands. The IRAP products are generated from two nearby retrotransposons using outward-facing primers. In REMAP, amplification between retrotransposons proximal to simple sequence repeats (microsatellites) produces the marker bands. Here, we describe protocols for the IRAP and REMAP techniques, including methods for PCR amplification with a single primer or with two primers and for agarose gel electrophoresis of the product using optimal electrophoresis buffers and conditions. This protocol can be completed in 1-2 d.     

与苹果Co基因紧密连锁的RAPD标记的筛选及其SCAR标记转换

以短枝富士(Spur Fuji)X舞姿(Telamon)的105株F1群体为试材,利用RAPD技术,结合集群分类分析法(BSA)进行了苹果柱型基因(Co)分子标记的研究。通过对300条随机引物的筛选,获得一个与Co基因紧密连锁的RAPD标记S1142682,连锁距离为2．86cM。对该标记片段进行序列测定,然后根据序列特点设计了4条特异引物(其中正向引物与反向引物各两条)。PCR结果显示,这4条引物的4种组合都可以扩增出柱型性状的特征带。选其中之一进行群体上的分析,结果表明该SCAR标记特征带与柱型性状的共分离行为与原RAPD标记表现一致。可见,此组合的引物可以作为该SCAR标记的特异引物。通过对S1142682标记片段序列分析发现,在 45～ 251区域含有一个可编码68个氨基酸残基的ORF。     

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples

PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.     

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Summary Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.     

Preliminary Report of RAPD Analysis in Paeonia suffruticosa Subsp. spontanea and Paeonia rockii

Ten different oligonucleotide primers of arbitrary sequence were used in Capillary and Air Thermocycler to amplify genomic total DNA of Paeonia suffruticosa subsp. spontanea and P. rockii isolated from several local populations in Shanxi, Shaanxi and Gansu provinces. Under the strictly standardized amplification condition for all the primers, these primers yielded clear and reproducible bands corresponding to amplified products and separable by agarose gel electrophoresis. Among a total of 71 bands amplified, 16(22. 5%) were polymorphic in a single individual of P. suffruticosa subsp. spontanea, while among a total of 76 bands 21(27.6%)were polymophic in a single individual of P. rockii. On an average,the pairwise marker difference between band profiles of conspecific individuals (different populations) was 7.9 for P. suffruticosa subsp. spontanea and 8.7 for P. rockii respectively. The average marker difference between P. suffruticosa subsp. spontanea and P. rockii was 10.3. Obviously, greater number of plants and primers will be required to detect satisfactorily level of genetic diversity. These preliminary results showed that intraspecific genetic diversity was low for the two endangered species. RAPD as a molecular marker was useful and feasible for detecting the genetic variation within species of wild moutans. And it was also potential for studying evolution and relationships between species.