随机扩增多态性对白色念珠菌分型的估价

利用随机扩增多态性（ＲＡＰＤ－ＰＣＲ）的方法对４８株白念珠菌Ｃａｎｄｉｄａ ａｌｂｉｃａｎｓ Ｂｅｒｋｈ进行了分析。由初步试验中,随机选用了１８种引物?筛选出ＯＰＡ－１４引物,该引物扩增的带型清晰可辨,不同菌株之间扩增的带数６－１２条不等,共有６条主带。扩增片段的长度粗略估计在３００－２０００ｂｐ左右。除个别菌株的带型相同以外,大多数菌株之间呈多态性分布,其带型数目和扩增的片段存在差异。对简单的Ｄ     

小麦双引物RAPD分析方法的研究

ＲＡＰＤ标记是近几年迅速发展的一种新型分子标记,标准的ＲＡＰＤ的反应是以１０个寡聚核苷酸作引物,通过ＰＣＲ反应扩增出基因组的部分片段,我们在研究外源ＤＮＡ导入小麦后外源遗传物质的追踪时,对这个方法进行了改进,采取了双引物进行扩增,结果双引物反应能够比单引物反应扩增出更多的多态性片段。分子杂交结果表明,双引物扩增出的新片段与单引物扩增片段无同源性,并对双引物扩增出的多态性片段产生的可能原因进行讨论。     

国内信息

ＤＥＧＣＭ技术中的ＲＤＡ、ＳＳＨ和ＲＳＤＤ差异表达基因分离技术 (ＤＥＧＣＭ )发展很快 ,主要是ＰＣＲ技术的发展和相应新技术的出现为ＤＥＧＣＭ注入了新的活力。ＰＣＲ引入差式筛选之中 ,可提高效力和降低假阳性率。若引入扣除杂交 ,可发展扣除扩增方案 ,并用特异引物结合于ＰＣＲ中选择扩增差异ｃＤＮＡ而形成代表性差式分析法 (ＲＤＡ)。采用抑制性ＰＣＲ选择性扩增差异基因片段 ,用ＤＮＡ链内“退火”优先于链间退火 ,使非目的序列产生“发夹”式互补结构 ,不能与引物配对 ,从而选择性抑制非目的基因片段的扩增 ,以此形成抑制性差…     

RAPD分析氮离子注入甜菊种子后的幼苗基因组DNA变异

应用ＲＡＰＤ 技术检测经低能氮离子注入甜菊纯系种子引起的幼苗基因组ＤＮＡ 变异。筛选出ＯＰＪ系列中的１５ 种引物对实验及对照基因组ＤＮＡ 进行了ＰＣＲ 扩增,共获扩增片段１０３ 条,分子量在０．３ － ３ｋｂ 之间,其中５ 种引物ＯＰＪ－ １ ,７ ,９,１１ ,１２ 扩增出差异片段１２ 条。结果表明,低能氮离子注入甜菊种子可引起体内基因组ＤＮＡ 发生突变;ＲＡＰＤ 技术是检测基因组ＤＮＡ 发生诱变的一种简便、有效方法。本文同时探讨了离子强度和Ｔａｇ ＤＮＡ 聚合酶用量对甜菊ＲＡＰＤ 分析结果的影响,以及氮离子注入诱变效应的可能机制。     

用分子生物学技术对草菇进行菌株鉴别

利用ＡＰ－ＰＣＲ和ＲＡＰＤ技术对三个草菇菌株进行鉴别,其结果与用草菇菌株Ｖ３４基因文库中的中等重复序列为探针进行限制性内切酶长度多态性分析（ＲＦＬＰ）,及对编码核糖体５．８ＳｒＲＮＡ的ＤＮＡ（ｒＤＮＡ）进行ＰＣＲ扩增后的产物进行限制性内切酶长主多态性分析（ＰＣＲ－ＲＦＬＰ）的结果相一致。这一结果显示出用这四种方法对草菇菌株进行鉴别具有相似的效果。同时用这四种方法构建的分子生物学标记显示出这三个菌株     

RAPD技术及其在微生物学方面的应用

198 0年 ,Ｂｏｔｓｅｉｎ提出ＤＮＡ限制性片段长度多态性 (ＲＦＬＰ)可以作为遗传标记 ,从此开创了直接应用ＤＮＡ多态的新阶段。 80年代后 ,ＤＮＡ多聚酶链式反应 (ＰＣＲ)的发展 ,使直接扩增ＤＮＡ的多态性成为可能 ,并在此基础上产生了许多种新型分子标记 ,诸如扩增片段多态性 (ＡＬＦＲ)、串联重复序列(ＶＮＴＲ)、单链构型多态性 (ＰＣＲ ＳＳＣＰ)、序列特异扩增区域 (ＳＣＡＲ)、随机扩增多态性ＤＮＡ(ＲＡＰＤ)等。而ＲＡＰＤ是较为突出的一种。ＲＡＰＤ是由Ｗｉｌｌｉａｍｓ和Ｗｅｌｓｈ在 1 990年各自独立发现的一种ＤＮＡ多态检…     

日本血吸虫中国大陆株基因多态性研究

对日本血吸虫中国大陆株湖南、湖北、江西、安徽、四川、云南隔离群以及一个实验室传代品系从基因水平进行了多态性研究。首先,在用ＰＣＲ－ＳＳＷＣＰ技术分析了２８Ｓ ｒＤＮＡ－Ｄ２高变区基础上,测定了该区安徽和云南隔离群的ＤＮＡ序列;其次,用ＰＣＲ获得了含有ＩＴＳ的ｒＤＮＡ片段,并对其进行了酶切点重复序列的多态性分析;最后,用ＲＡＰＤ技术分析了全基因组ＤＮＡ的多态性。结果表明,安徽与云南隔离群的２８Ｓ ｒ     

大鼠脑神经元特异性烯醇化酶基因的cDNA克隆及序列分析

用ＲＴ－ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑神经元特异性烯醇化酶的ｃＤＮＡ,将此包括编码全长ＮＳＥ４３３个氨基酸的ＤＮＡ片段重组入ｐＵＣ质粒,并用ＰＣＲ方法测定了全部顺序,经重复实验,发现Ｗｉｓｔａｒ大鼠与Ｆｏｒｓｓ－Ｐｅｔｔｅｒ报导的ＳＤ大鼠ＮＳＥ基因顺序,有两处单碱基的差别,其中一个涉及氨基酸的改变,同时还对ＲＮＡ的提取及长片段ＤＮＡ的ＲＴ－ＰＣＲ扩增进行了方法学的探讨。     

细胞内RT－PCR扩增免疫球蛋白重链可变区基因

通常,逆转录ＰＣＲ（ＲＴ－ＰＣＲ）需要高质量的ｍＲＮＡ,操作过程复杂,效率低且易受到ＲＮａｓｅ的破坏,为了简化操作,提高效率,用１０％甲醛盐溶液固定杂交瘤细胞,以ＮＰ－４０渗透化处理细胞后做ＲＴ－ＰＣＲ,获得了大约３５０ｂｐ的特异性免疫球蛋白重链可变区基因片段,与用同一对引物得到的常规ＲＴ－ＰＣＲ扩增产物一致。这项技术可用于获得特定结构基因片段,连结并扩增嵌合蛋白基因及构建多克隆免疫球蛋白文库等。     

RAPD在植物育种上的应用及其技术校正

ＲＡＰＤ(ＲａｎｄｏｍＡｍｐｌｉｆｉｅｄＰｏｌｙｍｏｒｐｈｉｃＤＮＡ) ,是在ＰＣＲ的基础上发展起来的一种ＤＮＡ多态性检测技术 ,由Ｗｉｌｌａｍｓ[1] 和Ｗｅｌｓｈ[2 ] 于 1990年建立 ,原理是以一系列不同的 ,少数碱基组成的随机核甘酸序列为引物 ,对样本基因组ＤＮＡ进行ＰＣＲ扩增 ,每个ＲＡＰＤ片段的产生要求在可增范围内存在与引物匹配的反向互补序列。引物结合位点ＤＮＡ序列的改变以及两扩增位点之间碱基的缺失插入或转换都能导致扩增片段的数目和长度的差异 ,经ＰＡＧＥ或琼脂糖凝胶电泳分离和ＥＢ染色来检测ＤＮＡ片段的多态…     

Using the fluorogenic 5' nuclease assay for high-throughput detection of (CA)n repeats in radiation hybrid mapping

Here, the power of the 5' nuclease assay to detect PCR products containing (CA)n repeats was compared with that of the classical electrophoretic analysis. This assay, which relies on the use of a unique (CA)10 energy transfer-labeled probe and the 5' nuclease activity of Taq DNA polymerase, was used to construct a dog radiation hybrid map consisting of microsatellite markers. Data from over 7000 PCRs were analyzed in parallel by the fluorogenic assay and the conventional ethidium bromide-stained, agarose gel-based assay. We show that the fluorogenic assay provides a sensitive, reliable and specific method for detecting (CA)n amplimers. Moreover, as no processing is required after the PCR, the risk of carryover contamination and the time required for sample analysis are greatly reduced. All radiation hyrid (RH) assays can be performed using a single PCR protocol, and a standard analysis method has been developed that enables numerically automated data processing. On the whole, using this strategy greatly enhanced the rapidity, throughput and accuracy of the RH mapping of microsatellite markers.     

Semiautomated clone verification by real-time PCR using molecular beacons

Conventional, high-throughput PCR analysis of common elements utilizing numerous primer sets and template DNA requires multiple rounds of PCR to ensure optimal conditions. Laborious gel electrophoresis and staining is then necessary to visualize amplification products. We propose novel multicolor molecular beacons, to establish a high-throughput, PCR-based sequence tagged site (STS) detection system that swiftly and accurately confirms marker content in template containing common repeat elements. A simple, one-tube, real-time PCR assay system was developed to specifically detect regions containing CA and GATA repeats. Ninety-six samples can be confirmed for marker content in a closed-tube format in 3 h, eliminating product confirmation on agarose gels and avoiding crossover contamination. Multiple STSs can be detected simultaneously in the same reaction tube by utilizing molecular beacons labeled with multicolor fluorophores. Template DNA from 260 RPCI-11 bacterial artificial chromosome (BAC) clones was examined for the presence of CA and/or GATA repeats using molecular beacon PCR and compared with conventional PCR results of the same clones. Of the 205 clones containing CA and GATA repeats, we were able to identify 129 clones (CA, n = 99; GATA, n = 30) by using molecular beacons and only 121 clones (CA, n = 92; GATA, n = 29) by conventional PCR amplification. As anticipated, 55 clones that contained sequences other than CA or GATA failed molecular beacon detection. Molecular beacon PCR, employing beacons specific for tandem repeat elements, provides a fast, accurate, and sensitive multiplex detection assay that will expedite verification of marker content in a multitude of template containing these repeats.     

Dynamics of vaginal bacterial communities in women developing bacterial vaginosis, candidiasis, or no infection, analyzed by PCR-denaturing gradient gel electrophoresis and real-time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

Genetic mapping of four dinucleotide repeat loci, DXS453, DXS458, DXS454, and DXS424, on the X chromosome using multiplex polymerase chain reaction.

Dinucleotide CA repeat sequences in the human genome have been shown to be highly polymorphic due to variation in the length of the repeat-containing segment. Therefore, these markers can serve as anchor loci in the construction of a high-resolution genetic map of the human genome. In this study, we improved the efficiency of typing dinucleotide repeats using multiplex polymerase chain reaction (PCR). Dinucleotide repeat sequences of four previously identified markers (DXS453, DXS458, DXS454, and DXS424) on the long arm of the X chromosome were simultaneously amplified in a single PCR reaction. This multiplex PCR was applied to genotype individuals from the 40 CEPH reference families, and the genotypic data were used to determine the map position of the four loci with respect to eight reference markers in the Xq region by linkage analysis.     

Dynamics of Vaginal Bacterial Communities in Women Developing Bacterial Vaginosis, Candidiasis, or No Infection, Analyzed by PCR-Denaturing Gradient Gel Electrophoresis and Real-Time PCR

The microbial flora of the vagina plays a major role in preventing genital infections, including bacterial vaginosis (BV) and candidiasis (CA). An integrated approach based on PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and real-time PCR was used to study the structure and dynamics of bacterial communities in vaginal fluids of healthy women and patients developing BV and CA. Universal eubacterial primers and Lactobacillus genus-specific primers, both targeted at 16S rRNA genes, were used in DGGE and real-time PCR analysis, respectively. The DGGE profiles revealed that the vaginal flora was dominated by Lactobacillus species under healthy conditions, whereas several potentially pathogenic bacteria were present in the flora of women with BV. Lactobacilli were the predominant bacterial population in the vagina for patients affected by CA, but changes in the composition of Lactobacillus species were observed. Real-time PCR analysis allowed the quantitative estimation of variations in lactobacilli associated with BV and CA diseases. A statistically significant decrease in the relative abundance of lactobacilli was found in vaginal fluids of patients with BV compared to the relative abundance of lactobacilli in the vaginal fluids of healthy women and patients with CA.     

鲤的微卫星引物对草鱼基因组分析适用性的初步研究

运用微卫星DNA-聚合酶链反应(STR—PCR)基因分型技术,选取已发表的28对鲤的微卫星引物,探讨鲤的引物用于草鱼基因组微卫星分析的可能性。通过优化PCR反应条件,消除了影子带和异源核酸双链分子两类微卫星相关假阳性带对STR—PCR分析的干扰。在此基础上,筛选出7对引物可在湘江野生草鱼基因组中扩增出特异性条带,占总数的25％;其中的4对引物(约占总数的14．3％)在8尾湘江野生草鱼小群体中即检测到了个体间等位基因的多态性。这些初步的结果表明鲤的微卫星引物可以用于草鱼基因组的分析。     

A polymorphic (CA)n repeat element maps the human glucokinase gene (GCK) to chromosome 7p.

A compound imperfect dinucleotide repeat element, [CA]4TTTGT[CT]7[CA]9AA[CA]4CCACATA[CA]3, was found approximately 10 kb 3' to the human glucokinase gene (GCK) from analysis of contiguous genomic DNA obtained from a bacteriophage lambda chromosome walk. Direct human genomic sequencing revealed the source of polymorphism to be variable numbers of CT and CA repeats. Altogether six alleles that range in length from +10 to -15 nucleotides compared to the most common (Z) allele have been identified. Alleles Z, Z + 2, and Z + 4 were present in American Blacks, Pima Indians, and Caucasians, with somewhat varied frequencies among the groups. Two alleles, Z + 10 and Z - 15, appear to be unique to American Blacks, while a Z + 6 allele was observed only in the Caucasian population studied. Observed heterozygosity of the polymorphism in the CEPH reference pedigree collection is 44% and the PIC 0.44. The polymorphism is assayed by PCR amplification and resolution of 32P-end-labeled products (ranging in length from 180 to 205 bp) on denaturing polyacrylamide sequencing gels. Using the PCR assay, the human glucokinase gene was physically localized to chromosome 7 in a panel of rodent/human somatic cell lines. Genetic analysis in CEPH pedigrees placed the dinucleotide repeat element, and thereby the human glucokinase gene, on chromosome 7p between TCRG and a RFLP locus D7S57. The glucokinase dinucleotide repeat genetic marker can now be used to assess the role of the glucokinase gene in diabetes by population association studies. In addition, this repeat marker and others flanking it on chromosome 7 can be used in linkage studies with families segregating the disorder.     

Type IV carbonic anhydrase is present in the gills of spiny dogfish (Squalus acanthias)

Physiological and biochemical studies have provided indirect evidence for a membrane-associated carbonic anhydrase (CA) isoform, similar to mammalian type IV CA, in the gills of dogfish (Squalus acanthias). This CA isoform is linked to the plasma membrane of gill epithelial cells by a glycosylphosphatidylinositol anchor and oriented toward the plasma, such that it can catalyze the dehydration of plasma HCO(3)(-) ions. The present study directly tested the hypothesis that CA IV is present in dogfish gills in a location amenable to catalyzing plasma HCO(3)(-) dehydration. Homology cloning techniques were used to assemble a 1,127 base pair cDNA that coded for a deduced protein of 306 amino acids. Phylogenetic analysis suggested that this protein was a type IV CA. For purposes of comparison, a second cDNA (1,107 base pairs) was cloned from dogfish blood; it encoded a deduced protein of 260 amino acids that was identified as a cytosolic CA through phylogenetic analysis. Using real-time PCR and in situ hybridization, mRNA expression for the dogfish type IV CA was detected in gill tissue and specifically localized to pillar cells and branchial epithelial cells that flanked the pillar cells. Immunohistochemistry using a polyclonal antibody raised against rainbow trout type IV CA revealed a similar pattern of CA IV immunoreactivity and demonstrated a limited degree of colocalization with Na(+)-K(+)-ATPase immunoreactivity. The presence and localization of a type IV CA isoform in the gills of dogfish is consistent with the hypothesis that branchial membrane-bound CA with an extracellular orientation contributes to CO(2) excretion in dogfish by catalyzing the dehydration of plasma HCO(3)(-) ions.     

Splicing together different regions of a gene by modified polymerase chain reaction-based site-directed mutagenesis

A modified polymerase chain reaction (PCR)-based site-directed mutagenesis method used to splice together different regions of a gene by deleting hundreds of nucleotides of undesired sequences is described. This method was inspired by a PCR-based site-directed mutagenesis method developed by Stratagene (La Jolla, CA, USA); the procedure and primer design were modified to enable the method to generate deletions several hundreds of nucleotides in length with an efficiency of 80-100%, and to delete two DNA fragments simultaneously in a single PCR. This method should be useful for deletion of large DNA fragments from a gene.     

含非竞争性内标四重荧光RT-PCR方法检测手足口肠道病毒方法的研究

旨在了解手足口病的流行和感染情况,并进行快速准确的检测。建立了含非竞争性内标的同时检测肠道病毒通用型、肠道病毒EV71型及柯萨奇病毒CA16型的四重荧光RT-PCR方法,对该方法的特异性、灵敏度等进行评价,并对多份临床样本进行应用检测。结果表明,该检测方法特异性强,对肠道病毒及其他人类非肠道病毒进行检测,显示了良好的特异性;该检测方法对EV71型和CA16型的检测灵敏度分别达到31.25 TCID50和1.25×102TCID50;将浓度为1×104TCID50及5×102TCID50的EV71样本进行重复性试验,其变异系数均小于1.5%;将浓度为5×102-5×105TCID50的EV71和CA16样本进行线性试验,其相关系数R2值在0.982-0.998之间。采用本研究建立的方法检测40份疑似临床样本,最后检出31份肠道病毒阳性样本,其中8例EV71型阳性,13例CA16阳性。另外,试验数据表明,内标对监控PCR抑制物的存在具有重要作用。本方法能同时快速检测所有肠道病毒并进行EV71型及CA16型的分型,并且灵敏度高、特异性好、扩增效率高,由于加入了内标,能有效地监控假阴性的出现,适合于手足口病的临床检测。