纳米金增强复杂体系中PCR扩增低拷贝基因的反应特异性初步研究

目的：利用纳米金颗粒提高复杂体系基因组低拷贝基因PCR扩增的反应特异性。方法：首先,模拟复杂基因组扩增模式体系,以接近单拷贝的λDNA为模板,在PCR过程中加入纳米金颗粒,设计优化实验,以便模拟建立复杂基因组低拷贝目的基因PCR扩增的模式体系。随后,扩增人类基因组的疾病相关的低拷贝基因模板（如人基因组肿瘤坏死因子基因外显子1的380bp）,以检验纳米金优化增强PCR反应特异性的实际效果。结果：在复杂体系基因组的低拷贝基因PCR扩增中,纳米金颗粒能够较好地增强其PCR反应的特异性。结论：初步表明基于纳米金的纳米粒子PCR方法可以对复杂的实际基因组体系低拷贝基因的PCR扩增起到优化作用,这对于PCR反应优化方法的改进、推广具有重要的参考价值。     

荧光标记DNA扩增片段长度多态性方法的改进

采用常规PCR试剂和合成的接头和引物,其中MseⅠ引物为荧光标记物FAM标记,并对扩增片段长度多态性(AFLP)程序进行了改进,优化了PCR反应和电泳条件,建立了一个新的、有效的反应体系,降低了实验费用,经比较实验结果与AFLP荧光标记试剂盒的实验效果一致.     

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.     

高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

溶藻弧菌中类霍乱弧菌毒力岛转座酶基因及侧翼序列分子生物学特征分析

[目的]调查类似霍乱弧菌毒力岛(VPI)转座酶(vpiT)的基因是否在溶藻弧菌中分布,并了解其全序列及侧翼序列的分子生物学特征.[方法]对94株溶藻弧菌是否携带类似VPI的vpiT基因进行PCR检测,对阳性株进行了PCR产物直接测序,根据获得的部分已知序列,设计引物,通过反向PCR扩增出全长类似vpiT的基因valT及部分侧翼片段,对反向PCR产物进行克隆测序,然后对获得的valT及侧翼序列进行生物信息学分析.[结果]发现94株溶藻弧菌中只有从粤东对虾池水分离的2个株E06011、E0612在PCR检测中产生了预期扩增片段.测序表明两者序列(valT-S1)完全一致.根据反向PCR及克隆最终获得的溶藻弧菌E0601全长valT基因及部分侧翼序列valT-S3.对valT-S3生物信息学分析表明:valT是一个高度类似于霍乱弧菌毒力岛vpiT的转座酶基因.[结论]根据上述结果及相关文献,有理由相信valT基因及其侧翼片段是异源获得,霍乱弧菌VPI元件或整体很可能在包括溶藻弧菌在内的弧菌种间转移.     

反向PCR法扩增花粉特异表达基因PSG076启动子

PSG076基因是从奥地利小麦品种‘Ferdinand’中分离的花粉特异性表达基因,功能未知.为获得可用于小麦基因工程的花粉特异性启动子,采用优化的反向PCR法分离PSG076启动子,获得了起始密码子上游约1.4 kb的启动子序列.生物信息学分析显示,该启动子除含有与花粉特异性表达相关的调控元件AGAAA和GTGA外,还含有花粉特异性表达相关的数量元件AGGTCA和AAATGA,推测其为活性较强的花粉特异性启动子.实验中对反向PCR方法的优化可提高扩增侧翼未知序列的效率,特别适用于启动子序列的扩增.     

溶藻弧菌HY9901 AcrA基因克隆与序列分析

Touchdown PCR扩增溶藻弧菌HY9901 AcrA基因部分序列,得一460bp片段,再以反向PCR和巢式PCR联合扩增其侧翼序列,拼接得一由1101 nt组成,共编码366 aa的完整基因.该基因演绎的氨基酸序列与几种弧菌的同源性都比较高,与创伤弧菌YJ016、副溶血弧菌RIMD 2210633、灿烂弧菌12B01、霍乱弧菌O1 N16961同源性分别为76%、73%、71%和70%.     

一个简便的DNA改组(DNA shuffling)操作程序

介绍了一个简便的DNA改组操作程序.首先利用PCR扩增了两段具有高度序列同源性的1 700 bp左右的基因片段,两者相比较同源性大于93%.然后将其等量混合后,在Mg²⁺存在的条件下,用DNaseⅠ切割成10～50 bp的小片段.这些小片段在不外加引物的前提下,利用PCR反应进行重聚,再将重聚物经过两轮正常的PCR扩增,获得了与原来片段大小相当的基因片段.这一技术有利于从一组序列同源性程度较高的基因库构建随机嵌合基因.     

4种快速PCR检测试剂法医学应用的初步研究

采用快速PCR扩增,探索其法医学应用价值.将AmpFLSTRIdentifiler试剂盒分别与4种不同的快速PCR检测试剂联合构建快速PCR体系,以9947A为模板,采用各自优化的循环参数进行扩增,并将各分型结果与常规方法进行比较,结果表明:联合构建的4种快速PCR体系均可获得与常规方法一致的DNA分型结果,扩增用时最短可减少至22 min.可见应用快速PCR方法进行扩增,可获得与常规方法一致的STR分型,并且显著缩短扩增时间,提高DNA分型速率.     

四引物扩增受阻突变体系PCR技术及其在动植物遗传育种研究中的应用

四引物扩增受阻突变体系PCR(Tetra-primer amplification refractory mutation system PCR,Tetra-primer ARMS PCR)技术是一种在普通PCR基础上发展起来的单核苷酸多态性(SNP)分型技术。该项技术综合了扩增受阻突变体系(Amplification refractory mutation system,ARMS)和四引物PCR(tetra-primer PCR)技术的优点,是对等位基因特异性PCR法的改良。它具有操作简便、分型快速、费用低廉等特点,在国内外生命科学领域尤其是遗传育种领域的应用越来越广泛。本文介绍了四引物扩增受阻突变体系PCR的技术原理及优势、结果检测手段和反应体系改进方法,并在此基础上对该技术在遗传育种研究中的应用进行综述。     

Characterization of the beta2-microglobulin gene of the horse

A clone containing beta(2)-microglobulin (beta(2)-m), the light chain of the major histocompatibility complex class I cell surface molecule, was isolated from an equine bacterial artificial chromosome library. This clone was used as a template for polymerase chain reaction (PCR) and unidirectional sequencing to elucidate the genomic sequence and intron/exon boundaries. We obtained 7,000 bases of sequence, extending from 1,100 nucleotides (nt) upstream of the coding region start through 1,698 nt downstream of the stop codon. The sequence contained regulatory elements in the region upstream of the coding sequence similar to those of the beta(2)-m gene of other species. The beta(2)-m gene was localized to horse chromosome ECA1q23-q25 by fluorescent in situ hybridization. This was confirmed by synteny mapping on a (horse x mouse) somatic cell hybrid panel. The sequence and intron/exon boundaries determined were used to design PCR primers to amplify and sequence the coding region of the beta(2)-m gene in other equids, including five breeds of domestic horse, one Przewalski's horse, five domestic donkeys and five zebras. A high degree of conservation was found among equids, illustrated by >98% (349/354) identity at the nucleotide level and 95% (113/118) at the amino acid level, because of non-synonymous nucleotide substitutions. The promoter detected in the region upstream of the coding sequence was subcloned and used in chloramphenicol acetyl transferase (CAT) assays to demonstrate the presence of a functional promoter. This study provides tools for the analysis of regulation of not only the horse beta(2)-m gene, but also for any genes dependent upon beta(2)-m for expression.     

Cloning and sequencing of the structural gene for the porin protein of Bordetella pertussis

Bordetella pertussis produces a porin protein which is a prominent outer membrane component found in both virulent and avirulent strains. N-terminal amino acid analysis of purified B. pertussis porin was performed and this amino acid sequence was used to design an oligonucleotide that was then utilized to screen a lambda gt11 library containing randomly sheared fragments of DNA from B. pertussis strain 347. One clone, lambda BpPor, was identified and subcloned into pUC18. A portion of the DNA insert in this subclone, pBpPor1, was sequenced and shown to contain the N-terminal region of the structural porin gene. This truncated gene sequence was used to design an additional oligonucleotide that was used to identify a clone, pBpPor2, which overlapped with pBpPor1 and contained a termination codon. The structural gene deduced from this sequence would encode a 365-amino-acid polypeptide with a predicted mass of 39,103 daltons. The predicted product also contains a signal sequence of 20 residues that is similar to that found in other porin genes. The predicted B. pertussis porin protein sequence contains regions that are homologous to regions found in porins expressed by Neisseria species and Escherichia coli, including the presence of phenylalanine as the carboxy-terminal amino acid. DNA hybridization studies indicated that both virulent and avirulent strains of B. pertussis contain only one copy of this gene and that Bordetella bronchiseptica and Bordetella parapertussis contain a similar gene.     

Genetics of the Tubulin Gene Families of Physarum

The organization of the alpha- and beta-tubulin gene families in Physarum was investigated by Mendelian analysis. Restriction endonuclease-generated DNA fragments homologous to alpha- and beta-tubulin show length polymorphisms that can be used as markers for genetic mapping. Analysis of meiotic assortment among progeny of heterozygotes allowed alpha- and beta-tubulin sequence loci to be defined. There are four unlinked alpha-tubulin sequence loci (altA, altB, altC and altD) and at least three unlinked beta-tubulin sequence loci (betA, betB and betC). The alpha-tubulin loci are not linked to the beta-tubulin loci. --Segregation of tubulin sequence loci with respect to ben mutations that confer resistance to antitubulin benzimidazole drugs was used to investigate whether any members of the alpha- or beta-tubulin gene families are allelic to ben loci. The beta-tubulin sequence locus betB is allelic to the resistance locus benD, the betA locus is probably allelic to benA and the alpha-tubulin sequence locus altC may be allelic to benC. The molecular implications of benzimidazole resistance phenotypes when only one of the expressed beta-tubulin gene family members mutates to drug resistance are discussed in relation to tubulin function.     

Mapping multiple co-sequenced T-DNA integration sites within the Arabidopsis genome

MOTIVATION: Insertion mutagenesis, using transgenes or endogenous transposons, is a popular method for generating null mutations (knockouts) in model organisms. Insertions are mapped to specific genes by amplifying (via TAIL-PCR) and sequencing genomic regions flanking the inserted DNA. The presence of multiple TAIL-PCR templates in one sequencing reaction results in chimeric sequence of intermittently low quality. Standard processing of this sequence by applying Phred quality requirements results in loss of informative sequence, whereas not trimming low-quality sequence causes inclusion of low-complexity homopolymers from the ends of sequence runs. Accurate mapping of the flanking sequences is complicated by the presence of gene families. RESULTS: Methods for extracting informative regions from sequence traces obtained by sequencing multiple TAIL-PCR fragments in a single reaction are described. The completely sequenced Arabidopsis genome was used to identify informative TAIL-PCR sequence regions. Methods were devised to define and select high quality matches and precisely map each insert to the correct genome location. These methods were used to analyze sequence of TAIL-PCR-amplified flanking regions of the inserts from individual plants in a T-DNA-mutagenized population of Arabidopsis thaliana, and are applicable to similar situations where a reference genome can be used to extract information from poor-quality sequence.     

Phylogenetic position of the spirochetal genus Cristispira.

Comparative sequence analysis of 16S rRNA genes was used to determine the phylogenetic relationship of the genus Cristispira to other spirochetes. Since Cristispira organisms cannot presently be grown in vitro, 16S rRNA genes were amplified directly from bacterial DNA isolated from Cristispira cell-laden crystalline styles of the oyster Crassostrea virginica. The amplified products were then cloned into Escherichia coli plasmids. Sequence comparisons of the gene coding for 16S rRNA (rDNA) insert of one clone, designated CP1, indicated that it was spirochetal. The sequence of the 16S rDNA insert of another clone was mycoplasmal. The CP1 sequence possessed most of the individual base signatures that are unique to 16S rRNA (or rDNA) sequences of known spirochetes. CP1 branched deeply among other spirochetal genera within the family Spirochaetaceae, and accordingly, it represents a separate genus within this family. A fluorescently labeled DNA probe designed from the CP1 sequence was used for in situ hybridization experiments to verify that the sequence obtained was derived from the observed Cristispira cells.     

cDNA cloning of porcine brain prolyl endopeptidase and identification of the active-site seryl residue

Prolyl endopeptidase is a cytoplasmic serine protease. The enzyme was purified from porcine kidney, and oligonucleotides based on peptide sequences from this protein were used to isolate a cDNA clone from a porcine brain library. This clone contained the complete coding sequence of prolyl endopeptidase and encoded a polypeptide with a molecular mass of 80,751 Da. The deduced amino acid sequence of prolyl endopeptidase showed no sequence homology with other known serine proteases. [3H]Diisopropyl fluorophosphate was used to identify the active-site serine of prolyl endopeptidase. One labeled peptide was isolated and sequenced. The sequence surrounding the active-site serine was Asn-Gly-Gly-Ser-Asn-Gly-Gly. This sequence is different from the active-site sequences of other known serine proteases. This difference and the lack of overall homology with the known families of serine proteases suggest that prolyl endopeptidase represents a new type of serine protease.     

Bacillopeptidase F of Bacillus subtilis: purification of the protein and cloning of the gene.

We have purified a minor extracellular serine protease from Bacillus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.     

Using the chicken genome sequence in the development and mapping of genetic markers in the turkey (Meleagris gallopavo)

The efficacy of employing the chicken genome sequence in developing genetic markers and in mapping the turkey genome was studied. Eighty previously uncharacterized microsatellite markers were identified for the turkey using BLAST alignment to the chicken genome. The chicken sequence was then used to develop primers for polymerase chain reaction where the turkey sequence was either unavailable or insufficient. A total of 78 primer sets were tested for amplification and polymorphism in the turkey, and informative markers were genetically mapped. Sixty-five (83%) amplified turkey genomic DNA, and 33 (42%) were polymorphic in the University of Minnesota/Nicholas Turkey Breeding Farms mapping families. All but one marker genetically mapped to the position predicted from the chicken genome sequence. These results demonstrate the usefulness of the chicken sequence for the development of genomic resources in other avian species.     

Multilocus sequence analysis of the actinobacterial genus Kribbella

Multilocus sequence analysis (MLSA) was used to refine the phylogenetic analysis of the genus Kribbella, which currently contains 17 species with validly-published names. Sequences were obtained for the 16S rRNA, gyrB, rpoB, recA, relA and atpD genes for 16 of the 17 type strains of the genus plus seven non-type strains. A five-gene concatenated sequence of 4099 nt was used to examine the phylogenetic relationships between the species of the genus Kribbella. Using the concatenated sequence of the gyrB-rpoB-recA-relA and atpD genes, most Kribbella type strains can be distinguished by a genetic distance of >0.04. Each single-gene tree had an overall topology similar to that of the concatenated sequence tree. The single-gene relA tree, used here for the first time in MLSA of actinobacteria, had good bootstrap support, comparable to the rpoB and atpD gene trees, which had topologies closest to that of the concatenated sequence tree. This illustrates that relA is a useful addition in MLSA studies of the genus Kribbella. We propose that concatenated gyrB-rpoB-recA-relA-atpD gene sequences be used for examining the phylogenetic relationships within the genus Kribbella and for determining the closest phylogenetic relatives to be used for taxonomic comparisons.