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.     

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。     

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.     

食用菌SSR序列开发策略研究进展

SSR序列开发策略主要有3大类:传统的基因文库筛选法、GenBank筛选法和富集文库筛选法。文中在对传统的基因文库筛选法、GenBank筛选法的优缺点及在食用菌SSR分子标记的开发应用进行综述的基础上,重点综述了富集文库筛选法,即基于RAPD技术的SSR开发策略、SSR兼并引物法、序列标签法构建SSR富集文库、vectorette PCR染色体步移法分离SSR位点、Suppression PCR染色体步移法分离SSR位点、引物延伸法构建SSR富集文库以及SSR杂交捕获法构建SSR富集文库的具体设计原理和步骤,并提出食用菌中SSR分子标识的开发现状和展望。     

侧翼序列获取技术研究进展

侧翼序列是指染色体中特定位点两侧的DNA序列,包含着候选基因、转录调控、染色体结构、生物安全等信息,在基因组学研究中具有重要的作用。侧翼序列获取技术主要应用于启动子和增强子等调控序列的克隆、鉴定T-DNA或转座子插入位点、染色体步移、全基因组空隙填补等,是结构基因组研究以及功能基因组研究的重要手段,在转基因动植物鉴定及安全管理等方面具有重要应用。随着分子生物学的发展,目前已经建立了许多侧翼序列的获取方法,依据技术原理可以分为质粒拯救法、反向PCR法、外源接头介导PCR法、半随机引物PCR法和基因组重测序法等5大类。本文系统总结了近年来侧翼序列获取技术的研究进展,并对这些技术的原理以及应用情况进行了较为系统的综述,为侧翼序列信息的获取提供参考。     

基因组步移法克隆小菜蛾CYP9G2基因的上游序列

利用4种产生平端切头的限制性内切酶消化小菜蛾(Plutella xylostella)的基因组DNA,然后利用DNA连接酶的催化作用,在4种不同平端切头的小菜蛾基因组DNA上连接一个氨基化的基因组步移衔接头序列,针对衔接头及已克隆的CYP9G2基因的序列,设计两对PCR上、下游引物,进行PCR扩增、T-A克隆和阳性克隆的巢式PCR验证,通过测序克隆到了小菜蛾CYP9G2基因上游未知序列约1.8 kb.通过对该基因的上游序列进行信息分析,发现1个可能的节肢动物动物转录起始子(Inr),3个CAAT样盒及1个抗氧化剂样反应因子,共5个可能的顺式调控元件.研究还表明,利用基因组步移方法可以快速地克隆已知序列的上游未知序列,实验操作经济、简便,对于已知cDNA序列或部分基因组序列的基因,其上游调控序列的克隆,基因组步移具有较高的实用价值.     

无缝克隆技术的研究进展

DNA克隆技术是分子生物学和基因工程研究必备的工具之一,但传统的以限制性内切酶酶切/连接为手段的克隆技术存在依赖限制性内切酶、连接效率低、耗时长和引入额外序列等缺点。近年来,无缝克隆技术发展迅猛,大量商业化克隆试剂盒也趋于成熟并在实验室被广泛应用,有力推动了蛋白质工程以及合成生物学的发展。本文从无缝克隆原理出发,对近年来发展出来的基于外切酶、PCR技术、同源重组、热稳定的连接酶等原理的无缝克隆技术进行了分类和讨论。特别是新酶资源的不断发现,如核酸外切酶XthA、FnCas12a突变体,与现有技术的相互融合,如Rec/ET重组和核酸外切酶共同使用,将有力推动无缝克隆技术的发展。     

小金海棠MxYSL5启动子克隆技术的比较

分别利用3种基于PCR技术的染色体步移法克隆了小金海棠MxYLS5基因的启动子,并比较了这三个体系的扩增产物。3种体系都可以获得特异性扩增产物,但扩增条带的长度和数量均不同,其中利用Genome Walking Kit扩增出来的条带特异性较好长度最长,而且特异性引物和简并引物的设计及退火温度的设置对3种体系的扩增产物起着关键的作用。基于PCR技术的染色体步移法为小金海棠MxYLS5基因启动子的克隆提供了一个稳定可靠的技术手段。     

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。     

L─山梨酮脱氢酶基因在大肠杆菌中的克隆和表达

参照已知的L－山梨酮脱氢酶基因序列,合成了两个引物序列,以AcetobacterliqueficiensIF012258的染色体DNA为模板进行PCR反应,克隆得到了L－山梨酮脱氢酶基因,经酶切验证与预期结果相同,序列测定结果也与已知序列一致。采用PCR方法在此基因的两端加上了E．coRI和HindⅢ两个酶切位点,经E．coRI和HindⅢ酶切后去掉了两端的多余序列后,将此片段连接到pKKH上,经诱导无蛋白表达,采用RcaI酶切启动子端,对载体pKKH则采用Mcol酶切,使表达载体的SD序列     

T-linker-specific ligation PCR (T-linker PCR): an advanced PCR technique for chromosome walking or for isolation of tagged DNA ends

Dozens of PCR-based methods are available for chromosome walking from a known sequence to an unknown region. These methods are of three types: inverse PCR, ligation-mediated PCR and randomly primed PCR. However, none of them has been generally applied for this purpose, because they are either difficult or inefficient. Here we describe a simple and efficient PCR strategy—T-linker-specific ligation PCR (T-linker PCR) for gene or chromosome walking. The strategy amplifies the template molecules in three steps. First, genomic DNA is digested with 3′ overhang enzymes. Secondly, primed by a specific primer, a strand of the target molecule is replicated by Taq DNA polymerase and a single A tail is generated on the 3′ unknown end of the target molecule, and then a 3′ overhang-T linker (named T-linker) is specifically ligated onto the target. Thirdly, the target is amplified by two rounds of nested PCR with specific primers and T-linker primers. T-linker PCR significantly improves the existing PCR methods for walking because it uses specific T/A ligation instead of arbitrary ligation or random annealing. To show the feasibility and efficiency of T-linker PCR, we have exploited this method to identify vector DNA or T-DNA insertions in transgenic plants.     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

A PCR-based marker for targeting small rye segments in wheat background

We attempted to develop a PCR-based marker that detects various segments of rye chromosome incorporated into wheat. We designed three sets of PCR primers based on the nucleotide sequence data of a rye repetitive sequence previously reported. One of the primer sets amplified a clear ca. 1.4 kb fragment in a rye cultivar but not in any form of wheat, diploid, tetraploid or hexaploid. We used this critical primer set for PCR of various wild species and cultivars of rye, an array of wheat plants carrying different rye chromosomes or small segments from different regions of rye chromosome 1R, and plants carrying parts of the rye B chromosome. The PCR amplified the 1.4 kb fragment in all the plant materials examined. We believe this PCR primer set will be useful as a universal PCR-based marker for the introgression of rye chromosome segments in the wheat genome.     

Characterization,genetic variation and chromosomal assignment to sheep chromosome 2 of the ovine heart fatty acid-binding protein gene (FABP3)

The purpose of this study was to locate and detect genetic variation in the sheep FABP3 gene, a candidate gene for milk and meat quality traits in sheep. We have obtained an almost complete sequence (4,689 bp, excluding a part of intron 1) of the sheep FABP3 gene using PCR-based comparative genome walking. Sheep FABP3 has been located to chromosome 2 by sheep sequence-specific PCR on DNA from a sheep/rodent cell hybrid panel, and confirmed by linkage mapping using the International Mapping Flock. Direct sequencing of PCR products amplified from different DNA samples of Manchega breed sheep over the complete sheep FABP3 gene revealed 13 SNPs, one CTC insertion/deletion and a variable polyA tract. This poly A tract was found in association with a SINE/artiodactyls repeat. In addition, two SNPs were screened in different sheep breeds.     

Optimized adaptor polymerase chain reaction method for efficient genomic walking

Genomic walking is one of the most useful approaches in genome-related research. Three kinds of PCR-based methods are available for this purpose. However, none of them has been generally applied because they are either insensitive or inefficient. Here we present an efficient PCR protocol, an optimized adaptor PCR method for genomic walking. Using a combination of a touchdown PCR program and a special adaptor, the optimized adaptor PCR protocol achieves high sensitivity with low background noise. By applying this protocol, the insertion sites of a gene trap mouse line and two gene promoters from the incompletely sequenced Xenopus laevis genome were successfully identified with high efficiency. The general application of this protocol in genomic walking was promising.     

Directional genome walking using PCR

We describe here a PCR-based "directional genome walking" protocol. The basic procedure for the amplification consists of two rounds of PCR. A primary PCR was performed, on the genomic DNA using a biotinylated primer specific to a known sequence in the genome along with four universal walker primers that were designed with partial degeneracy. The biotinylated primary PCR products were immobilized on streptavidin-linked paramagnetic beads. This step removed all nonspecific amplification products, and the purified template was used for the second PCR using a nested primer and the walker primer-2 to increase specificity. This technique is potentially useful for cloning promoter regions and has been successfully used to isolate 5'-flanking genomic regions of many cDNA clones previously isolated by us.     

A PCR-based approach to sequence the Candida tropicalis HSP90 gene

The heat shock protein 90 (hsp90) gene sequence is known to be highly conserved across the species barrier. A PCR-based method was thus utilised in an attempt to sequence the Candida tropicalis hsp90 gene. Primers for PCR were designed from conserved regions of the gene, which were identified by comparing the Saccharomyces cerevisiae and Candida albicans hsp90 gene sequences. Different sets of primers were designed to amplify and obtain overlapping DNA sequences of the C tropicalis gene. PCR was carried out on genomic DNA of Candidca tropicalis and the PCR products were cloned into suitable vector molecules for sequencing. In this way, a 2,070-basepair sequence of the C. tropicalis hsp90 gene was obtained. The PCR-based approach proved to be an easier method of obtain the sequence of a highly conserved gene, as compared to more conventional methods.     

The porcine gonadotropin-releasing hormone receptor gene (GNRHR): genomic organization, polymorphisms, and association with the number of corpora lutea.

The interaction of gonadotropin-releasing hormone (GNRH) and its receptor (GNRHR) is critical in the endocrine regulation of reproduction. The gene (GNRHR) encoding the receptor has been mapped to porcine chromosome 8. There is evidence for three quantitative trait loci (QTL) influencing ovulation rate on this chromosome. We obtained an almost complete sequence (3993 bp, excluding intron 1) of the porcine GNRHR gene using PCR-based comparative genomic walking and inverse genomic walking approaches. Twelve polymorphisms were detected by sequencing of pooled DNA of Chinese Taihu and European Large White pigs, including 7 base substitutions and 5 insertions-deletions (indels). A F2 population of Meishan x European Large White pigs was genotyped for a TG indel in the promoter region, and a C/G substitution in the 3' UTR (untranslated region). A significant association of the C/G substitution with number of corpora lutea at first parity was observed.