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1.
用PCR—RFLP方法研究藏族HLA0—DQA1和—DQB1基因多态性   总被引:4,自引:1,他引:3  
李霞  张咸宁 《遗传学报》1998,25(5):398-402
应用目前HLA研究领域中成熟的、有效的PCR-RFLP基因分型技术,从DNA水平对藏族健康群体进行了HLA-DQA1(49人)和-DQB1(49人)基因分型,这在国内外属首次。所采用的PCR-RFLP基因分型技术是在HLA-DQA1和-DQB1各等位基因全部序列已知的情况下,对其第2个外显子碱基序列扩增进而进行RFLP分析的方法。这种方法得到的RFLP的所有片段都是已知序列,因而精确度很高,同时为  相似文献   

2.
中国西北地区汉,回,维,藏民族HLA—DRB基因多态性的研究   总被引:29,自引:2,他引:27  
赖淑苹  任惠民 《遗传学报》1999,26(5):447-457
按照第11届国际相容性抗原研讨会工作会议HLAⅡ类PCR-SSO分型标准和美国国立骨髓供者计划组织对HLA DRB位点等位基因分型要求,设计合成1对引物,扩增HLA DRB DNA片段,长度为256bp,设计合成不同片段大小探针27种,可检出DRB座位上DRB1的39种等位基因,DRB3的3种等位基因,DRB4的1种等位基因和DRB5的34种等位基因。  相似文献   

3.
应用聚合酶链反应(PCR),对分离自不同地区、不同宿主来源的26株肾综合征出血热病毒进行了分型,其中包括4个型别的国际标准株,用异硫氰酸胍-酚-氯仿方法从感染的Vero-E6细胞中提取总RNA,设计了5对寡核苷酸引物,一对为汉坦病毒特异性引物;4对为不同型特异性引物。PCR分型表明,26株中除1株Rr可同时被汉坦(HYN)和Seoul(SEO)两型特异性引物扩增外,其余25株分别只被4个型别引物中的一个所扩增,依次为HTN16株,SEO7株,Puumala(PUU)1株,ProspectHill(PH)1株。PCR分型的结果与空斑减少中和试验完全一致,表明PCR可以对肾综合征出血热病毒准成分型。应用限制性内切酶分析了扩增产物,结果与理论基本一致,证实了扩增产物的特异性。  相似文献   

4.
病理性近视与HLA的关联性研究   总被引:3,自引:0,他引:3  
用PCR-RFLP方法对江浙沪籍汉族55例病理性近视眼(PM)患者的HLAⅡ类DQB1基因的第二个外显子进行了基因分型。结果发现HLA-DQB1*0201、*0303、*0401等位基因在PM患者中和正常人中的分布有显著的差异(Pc<0.05,AF分别为0.1636,0.1091,0.1636,0.1091vs.0.0400,0.0300,0.0400,0.0200),可能与PM的致病相关。DQB  相似文献   

5.
本文利用PCR技术建立一种对HSV直接基因分型的方法。在HSV-Ⅰ、Ⅱ两型的DNA多聚酶基因上设计了一条两型共同的上游引物(HDP-B)和两条型特异的下游引物(HDP-1、HDP-2)。三条引物共同组成一个扩增反应体系,在HSV-Ⅰ产生543bp条带,HSV-Ⅱ产生372bp条带,据此在基因水平上对HSV进行分型。5株不同来源的HSV(2株Ⅰ型,3株Ⅱ型)分型结果与病毒分离及血清学方法完全一致。该  相似文献   

6.
将遴选的经适当接尾的12个HLA-DQA1序列特异性寡核苷酸固定在一张滤膜上,用生物素标记的DQA1特异性扩增产物与滤膜上的序列特异性寡核苷酸在四甲基氯化铵杂交体系中杂交,然后经洗膜封膜,杂交信号用非放射性的碱性磷酸酶显色法检测,根据杂交斑点的显示结果分析标本的基因型。采用这种方法初步确定了HLA-DQA1位点8种单倍型等位基因:DQA10101、0102、0103、0201、03011、0401、0501和0601.非放射性反相杂交法可对各种来源的杂合性标本进行HLA-Ⅱ类基因快速分型,并适合在临床器官移植的组织分型配型、疾病易感性研究和法医鉴定等领城中应用。  相似文献   

7.
近几年,有关幽门螺杆菌(HP)基因分型方法及其应用的研究取得了很大进展。基因分型方法包括:质粒分型、限制性内切酶分型(REA)、核糖分型、染色体DNA脉冲场凝胶电泳(PFGE)分型、多聚酶链反应限制性内切酶消化(PCR-RFLP)分型、任意引物PCR(AP-PCR)分型、PCR单链构型多态性(PCR-SSCP)分型和核苷酸序列分析等。基因分型方法广泛用于HP的研究,如基因图的构建、感染复发、耐药机  相似文献   

8.
用PCR方法从pPAIJ.7中扩增人纤溶酶原激活剂抑制物2型(PAI-2)基因,与pPUC18重组,经限制性内切酶片段分析与核苷酸序列分析,获得全长人PAI-2基因.PAI-2基因与表达载体pPIC9重组,构建受乙醇氧化酶1基因(AOX1)启动子与转录终止区控制的酵母表达质粒,转化GS115宿主菌,经表型筛选和PCR扩增筛选阳性克隆,用甲醇诱导表达,重组PAI-2以分泌型表达,占分泌总蛋白的30%,具PAI-2抗原性,与低分子量尿激酶形成了抗SDS复合物,具抑制纤溶的活性(91.4AIU/ml).对培养条件也进行了探讨.  相似文献   

9.
用抗单纯疱疹病毒(HSV)型共同性gC和gD单克隆抗体(McAb),包被Eppendorf管,捕捉HSV,同时加入3个引物:一个是HSV-1/HSV-型共同性上游引物,另两个分别是HSV-1和HSV-2型特异性下游引物。借此建立了能直接分型检测HSV的抗原捕获聚合酶链式反应(AC-PCR)。HSV-1的扩增产物为477bp,HSV-2的为399bp,两型病毒经AC-PCR扩增后产生分子量不同的DN  相似文献   

10.
本实验用人重组r-干扰素(rhu-IFN)作用HEP-2细胞后HLA-DR抗原和增殖细胞核抗原(PCNA)表达的检测来探讨r-干扰素对HEP-2细胞HLA-DR抗原表达诱导作用及体外抗增殖活性。用单克隆抗体CR3/43(抗HLA-DR)和Ki-67(抗PCNA)。以链霉素一生物素技术(LSAB)检测HEP-2细胞HLA-DR抗原和PCNA表达,结果显示:r-IEN诱导HLA-DR抗原和抑制PCNA表达其强弱与r-IFN剂量有关。资料提示:r-IFN不仅对HEP-2细胞有细胞毒作用,同时能调节其细胞膜特性,因而在喉癌的治疗中是有效的。  相似文献   

11.
Evaluation of Cryptosporidium parvum genotyping techniques.   总被引:7,自引:0,他引:7  
We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.  相似文献   

12.
红鳍东方鲀(Takifugu rubripes)MC4R基因的多态性分析   总被引:1,自引:0,他引:1  
采用PCR-SSCP(single strand conformation polymorphism)技术和DNA测序方法分析红鳍东方鲀MC4R(Melanocortin-4receptor)基因编码区多态性。在MC4R基因编码区48 nt和264 nt均发生了碱基的转换突变(G→A),两个突变位点分别位于M1和M2引物扩增产物中。引物M1扩增产物SSCP分析得到两种基因型:AA基因型和AB基因型,并且AA基因型和A等位基因频率明显高于AB基因型和B等位基因。引物M2扩增产物也得到两种基因型:CC基因型和CD基因型,CC基因型和C等位基因频率明显高于CD基因型和D等位基因。遗传变异结果分析表明,两个突变位点均属于低度多态性,而且群体遗传杂合度较低,反映了该群体的遗传一致性较高。  相似文献   

13.
Evaluation of Cryptosporidium parvum Genotyping Techniques   总被引:1,自引:0,他引:1       下载免费PDF全文
We evaluated the specificity and sensitivity of 11 previously described species differentiation and genotyping PCR protocols for detection of Cryptosporidium parasites. Genomic DNA from three species of Cryptosporidium parasites (genotype 1 and genotype 2 of C. parvum, C. muris, and C. serpentis), two Eimeria species (E. neischulzi and E. papillata), and Giardia duodenalis were used to evaluate the specificity of primers. Furthermore, the sensitivity of the genotyping primers was tested by using genomic DNA isolated from known numbers of oocysts obtained from a genotype 2 C. parvum isolate. PCR amplification was repeated at least three times with all of the primer pairs. Of the 11 protocols studied, 10 amplified C. parvum genotypes 1 and 2, and the expected fragment sizes were obtained. Our results indicate that two species-differentiating protocols are not Cryptosporidium specific, as the primers used in these protocols also amplified the DNA of Eimeria species. The sensitivity studies revealed that two nested PCR-restriction fragment length polymorphism (RFLP) protocols based on the small-subunit rRNA and dihydrofolate reductase genes are more sensitive than single-round PCR or PCR-RFLP protocols.  相似文献   

14.
A DD genotype of the angiotensin I-converting enzyme gene has been implicated in various diseases. However, genotype frequencies differ between previous reports, and data on the association of DD genotype with disease are sometimes conflicting. Although elimination of mistyping is of crucial importance, assessment of the accuracy of currently adopted typing methods has rarely been performed. Mistyping of the DD genotype is reported to occur by a conventional method with insertion/ deletion (I/D) flanking primers using polymerase chain reaction (PCR). We investigated whether currently adopted genotyping methods by PCR are reliable or not. We genotyped 248 patients by conventional PCR methods with I/D flanking primers with or without dimethyl sulfoxide (DMSO), and confirmed the DD genotype with insertion-specific primers with or without DMSO. Mistyping occurred frequently, not only in both methods without DMSO but also in a modified method with I/D flanking primers with inclusion of DMSO. Typing by these methods proved to lead to erroneous results more frequently than had been previously thought. To reduce mistyping frequency, initial PCR genotyping with I/D flanking primers with an inclusion of DMSO, followed by confirmation of the DD genotype by insertion-specific primers with DMSO, is recommended. Received: 15 August 1996 / Accepted: 6 March 1997  相似文献   

15.
人类血小板抗原1~6系统同步基因分型的研究   总被引:4,自引:1,他引:3  
邓志辉  吴国光  李大成 《遗传》2004,26(5):594-598
为研究采用PCR—SSP技术,建立可靠的人类血小板抗原HPA-1,2,3,4,5,6系统的同步基因分型方法,并以所建立的方法研究血小板抗原。设计合成18条序列特异性引物,探索最佳退火温度,通过调整引物浓度、Mg2+离子浓度,使HPA-1~6系统等位基因在同一条件下进行同步扩增和扩增产物在同一凝胶中进行同步电泳。引物的特异性和灵敏度采用基因型已知的质控DNA进行验证。应用此方法,对2000年度国际输血协会(ISBT)第十届血小板基因定型与血清学工作组送检的15份考核样本(其中血样2份,DNA样本13份)进行了基因分型。用此方法检测质控DNA,结果与已知的HPA基因型完全相符;15份第十届血小板基因定型与血清学工作组的考核样本的检测结果,与ISBT公布的结果完全相同,准确率达100%。Abstract: To set up the simultaneous genotyping of human platelet antigens of 1,2,3,4,5,6 system by PCR—SSP assay and use the genotyping method for the study of platelet antigens. In this study, 18 sequence-specific primers were designed and synthesized. The annealing temperature for all sequence-specific primer pair, the concentration of each primer pair and the concentration of Mg2+ were adjusted to the optimum so that HPA-1 to 6 systems could be amplified simultaneously under the same PCR cycling parameters. The electrophoresis of PCR products was conducted simultaneously on the same agarose gel. Control DNA samples that genotypes known were used to confirm the sensitivity and specificity of each sequence-specific primer. 15 coded samples (including 2 blood samples and 13 DNA samples) distributed by 10TH Platelet Genotyping and Serology Workshop of the International Society of Blood Transfusion (ISBT) were typed for HPA-1 to 6 systems by this method. A concordance rate of 100 percent was observed between the results of control DNA samples typed by our PCR—SSP assay and the data of known specificity of control DNA. The results of 15 coded samples tested by our method agreed well with the results provided by ISBT report.  相似文献   

16.
寡核苷酸DNA Microarray用于HLA DRB1基因分型的研究   总被引:18,自引:1,他引:17  
对寡核苷酸DNA Microarray用于HLA DRB1基因分型的技术进行研究。常规的酚/氯仿法提取标准血样基因组DNA,在DRB1的exon2区域设计一对引物,经PCR扩增基因组相应区段并用Cy5-dCTP进行标记。设计寡核苷酸分型探针,将探针固定在APS-PDC法制作的DNA Microarray上,用标记的PCR产物与之杂交,扫描仪对杂交效果进行扫描,Imagene软件对杂交图像进行分析。共检测了33例标准血样的HLA DRB1基因型。检测结果证明研制的DNA Microarray准确、灵敏。DNA Microarray技术可以有效地检测DRB1等位基因,对比常规的PCR-SSP和PCR-SSO方法、分型基因芯片方法更为直观,并有集成化优势。  相似文献   

17.
Several genes encoding different cytokines may play crucial roles in host susceptibility to lung cancer, since cytokine production capacity varies among individuals and depends on cytokine gene polymorphisms. The association between cytokine gene polymorphisms with primary lung carcinoma was investigated. DNA samples were obtained from a Turkish population of 44 patients with primary lung cancer, and 59 healthy control subjects. All genotyping (IFN-gamma, TGF-beta1, TNF-alpha, IL-6 and IL-10) experiments were performed using sequence-specific primers (SSP)-PCR. When compared to the healthy controls, the frequencies of high/intermediate producing genotypes of IL-10 and low producing genotype of TNF-alpha were significantly more common in the patient group. It is noteworthy that lung cancer patients with the TGF-beta T/T genotype in codon 10 had statistically longer survival compared to those having the C/C genotype (Kaplan-Meier survival function test, log rank significance = 0.014). These results suggest that IL-10, TNF-alpha and TGF-beta1 gene polymorphisms may affect host susceptibility to lung cancer and the outcome of the patients.  相似文献   

18.
目的:人类染色体是二倍体,这使得以测序方法研究多态性区域时会遇到不明确杂合子,这个问题在HLA-DPB1的分型上尤为突出。试图寻找一个来解决HLA-DPB1分型中不明确杂合子比例高给分型带来的困难的方案。方法:对946例样品进行HLA-DPB1分型,其中不明确杂合子有353例,共30种,占总例数的37%。建立了一套SSP分型方法,设计上游引物6条,下游引物15条,每一个样品同时用两对特异性引物进行扩增,两对引物分别代表两种不同的杂合模式。再从30种不明确杂合子类型中各挑2个样品进行克隆测序,结果与SSP分型结果比较。结果:SSP分型方法采用同一套扩增体系与两套循环反应条件,只需一次PCR扩增,就可以方便快捷地实现不明确杂合子的分辨,其结果与克隆测序结果相符。结论:与以往的克隆测序、SSCP方法相比,本研究中SSP分型方法具有高效率、高通量、省时省力的特点。  相似文献   

19.
乙型肝炎病毒(Hepatitis B virus,HBV)是引起急性和慢性肝炎的最主要的病原[1]。目前根据HBsAg的共同抗原决定簇“α”和两对相互排斥的抗原决定簇将HBV分为ayw1, ayw2, ayw3, ayw4,ayr, adw2, adw2, adw4, adrq 和adrq-9种不同的血清学亚型,1988年Okamoto[2]等根据HBV基因组核苷酸的差异又提出了HBV基因型的概念,并以全基因组核苷酸差异≥8%,定为基因型分型标准。目前从世界不同地区分离的乙型肝炎病毒分离株已被分为A、B、C、D、E、F、G、H等8种不同的基因型[3~5]。包括中国、日本和东南亚在内的亚洲地区主要流行B、C两种基因…  相似文献   

20.
Salvia miltiorrhiza (SM), a widely popular Chinese herb, is grown in various regions in China. Identifying SMs grown in different provinces of China is difficult, and therefore genotyping these collections would be highly valuable. Based on the techniques of sequence-related amplified polymorphism and target region amplified polymorphism, a novel PCR-based molecular marker technique called conserved region amplification polymorphism (CoRAP) is reported in this study to genotype SMs. The CoRAP technique is based on the use of two primers: fixed and arbitrary primers. The former is derived from target EST sequences deposited in Genbank; while, the core sequence (CACGC) of the latter is a conserved region found in most introns. In the present study, we utilized CoRAP to genotype SMs from different geographical origins. PCR amplification is performed for 30 cycles at an annealing temperature of 52°C. Each PCR reaction has generated as many as 30–50 fragments of 50 to 1,000 bp in size. The successful DNA genotyping of SMs by CoRAP was achieved. This new genotyping method is rapid, efficient, and reproducible.  相似文献   

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