连接酶介导的诱导荧光共振能量转移法检测基因点突变

发展了一种基于连接酶介导的诱导荧光共振能量转移技术用于基因点突变的准确快速检测方法.针对特定突变位点设计的核酸探针.当与模板之间完全匹配时,被连接形成一条长的双链,双链特异性嵌入荧光染料SYBR Green I插入新生的双链区域.诱导荧光共振能量转移发生.相反,核酸探针与模板之间不匹配,则不能诱导荧光共振能量转移的出现.利用该方法,成功实现了β地中海贫血遗传病两种普遍存在的点突变Ivs-2-654(C→T)和CD17(A→T)的基因型检测.     

三色荧光技术在SNP检测中的应用

利用三色荧光标记的A、C、T双脱氧核苷酸单碱基延伸的方法结合编码寡核苷酸芯片技术检测单核苷酸多态性 (SNP)的基因型。以beta地中海贫血样本基因 (HBB基因 )突变作为模型的研究结果显示该方法能同时对多位点的SNP进行检测。     

遗传性β地中海贫血病CD17点突变的快速简便检测

利用聚合酶延伸技术及双链特异性嵌入染料的特性,建立了一种快速简便的基因点突变的检测方法．在特定引物聚合过程中。不同基因。型的聚合反应进程被实时转换为荧光信号,通过监测荧光信号的变化实现基因点突变的快速检测,不需要复杂的凝胶电泳、荧光和同位素标记等操作．通过对卢珠蛋白基因CD17点突变的检测,证实该方法是一种廉价、简便、快速的筛查遗传性地中海贫血病卢珠蛋白基因CD17点突变的方法,该方法可扩展到各种基因的点突变检测．     

基于锁核酸及等位位点特异性扩增技术的KRAS基因突变检测荧光定量PCR方法研究

基于等位位点特异性扩增的原理,设计锁核酸修饰KRAS基因突变特异性扩增引物,结合封阻探针技术,建立检测KRAS基因突变的荧光定量PCR方法。结果发现,锁核酸修饰的引物及探针可显著提高等位位点特异性扩增技术用于复杂样本中的微量基因突变检测的敏感度,该技术检测KRAS基因突变的敏感性可达0.01%~0.1%。进一步用建立的荧光定量PCR方法检测52例结直肠癌患者血浆标本,并用DNA测序法作为对照,同时用健康人血浆标本建立阴性检测结果判读标准,以初步评价该方法应用于循环DNA中KRAS基因突变检测的可行性。结果发现结直肠癌患者KRAS基因突变主要是G12C、G12A和G12R,而且q PCR法的阳性检出率为46.15%,高于DNA测序法(13.46%),阴性结果与DNA测序法的符合率为100%。此外,结直肠癌患者外周血KRAS基因的突变检出率与文献报道组织标本中的突变检出率及常见突变类型基本相符。上述结果说明该方法检测循环肿瘤DNA(circulating tumor DNA,ct DNA)具有较高的可靠性,可以用于肿瘤患者循环血液中KRAS基因突变的检测。     

高分辨率熔解曲线法的非标记探针基因分型技术

PCR反应中利用荧光检测技术对已知位点进行基因分型时常采用荧光标记的寡核苷酸做探针。近年来新兴起的高分辨率熔解曲线技术可以采用非标记的探针对已知位点的SNP(single nucleotide polymorphism)或突变进行基因分型研究。采用非标记探针法对已知位点的基因分型研究具有廉价、快速、简便等特点,因此被大量应用在和疾病、形状等相关的一些多肽位点的研究中。本文较详细地介绍该技术的基本原理和实验中的注意事项。     

湖南地区人群中线粒体12S rRNA基因A1555G突变筛查

为建立快速、简便、准确筛查线粒体DNA 12S rRNA基因A1555G突变的基因检测技术平台,收集1 758例（女性808例,男性950例）正常人群样本,利用Bsm AⅠ酶切法筛查线粒体DNA A1555G突变以及通过实时荧光定量Taqman探针法和直接测序法对筛查结果进行验证,结果检测到2例A1555G阳性突变样本,其中1例为男性,1例为女性。实时荧光定量Taqman探针法与Bsm AⅠ酶切法、直接测序法检测结果完全相符,未发现假阳性和假阴性,该方法具有结果准确直观、简单省时,特异性强,敏感性高的优点,适用于对母系遗传性耳聋线粒体DNA A1555G突变的大规模筛查或氨基糖甙类抗生素应用前的预防性检测。     

应用LNA-PCR法检测乙型肝炎病毒阿德福韦酯耐药位点基因突变

目的:建立简便、快速、灵敏的锁核酸(locked nucleic acid,LNA)探针实时荧光聚合酶链反应(PCR)检测方法,检测乙型肝炎病毒(hepatitis B virus,HBV)阿德福韦酯(Adefovir dipivoxil,ADV)耐药相关位点(rtA181V、rtN236T)突变。方法:通过基因测序筛选阳性样本,进而构建ADV rt181和rt236位点野生株和突变株重组质粒,设计包含扩增阿德福韦酯rtA181V和rtN236T耐药位点在内的特异性引物和LNA荧光探针,以构建的重组质粒为标准品建立实时荧光PCR反应体系,并通过与基因测序平行检测血清样本以判断检测方法的可行性与准确性。结果:所建立的LNA-PCR法能够检测102copies/ml的HBV中ADV基因突变,同时具备较高的特异性。通过对89例ADV治疗一年后HBV阳性临床样本进行检测,有8例(8.98%)rtA181V突变、5例(5.61%)rtN236T突变、2例(2.24%)rtA181V和rtN236T混合突变,检测结果与测序结果一致。结论:所建立的LNA-PCR法是一种简便、快速、灵敏的基因突变检测方法,能有效的区分单碱基突变,对慢性乙型肝炎患者德福韦治疗过程中耐药突变的监控和抗病毒药物的调整具有指导意义。     

双链探针实时荧光PCR核酸检测新技术研究*

目的:采用一种“双链探针”实时荧光PCR技术,提高HBV核酸检测灵敏度,并在同一反应管中实现代谢酶CYP2C19^*2基因型检测。方法:采用双链探针与TaqMan探针同时检测不同浓度HBV血清样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和结果统计分析;采用双链探针检测代谢酶CYP2C19^*2不同基因型样本,使用上海宏石SLAN 96实时荧光PCR仪进行核酸Ct值检测和基因型确定。结果:不同浓度HBV血清样本检测,双链探针荧光本底低,检测灵敏度更高,与TaqMan探针检测结果相比,两者核酸检测Ct值存在显著性差异(P<0.05);双链探针检测36份样本的代谢酶CYP2C19^*2基因型,检测结果与Sanger测序结果完全一致。结论:双链探针实时荧光PCR检测技术可完成目的基因的高灵敏核酸检测,也可实现基因型分析。     

应用TaqMan探针荧光定量PCR技术鉴定Lepr~(db/+)小鼠子代基因型

目的建立一种高效的应用Taq Man探针荧光定量PCR技术对Lepr~(db/+)小鼠子代基因分型的方法。方法提取228例Lepr~(db/+)小鼠子代鼠尾DNA,针对Lepr基因的突变位点(rs1801133)设计1对PCR引物和2条Taq Man探针。设定条件进行实时荧光PCR扩增,用SDS软件对SNP位点进行分型。通过2月龄动物的肥胖表现型验证并进行Hardy-Weinberg平衡检验。结果用建立的Taq Man探针荧光定量PCR方法对228份样本进行检测,其中GG基因型64份,基因型频率为0.1929;GT基因型123份,基因型频率为0.5395;TT基因型41份基因型频率为0.2807。Taq Man探针荧光定量PCR方法分型结果与通过肥胖表现型分型结果比较,灵敏度为97.56%,特异度为99.47%。结论应用Taq Man探针荧光定量PCR技术可实现对Lepr~(db/+)小鼠子代基因位点的早期分型检测,方法简便,高效。     

肥厚型心肌病致病基因热点突变位点Taqman-MGB探针检测方法的建立

肥厚型心肌病(hypertrophic cardiomyopathy,HCM)的分子遗传学基础为基因突变。本研究旨在建立一种用于HCM致病相关基因热点突变位点的Taqman-MGB实时荧光PCR检测方法。以HCM患者基因组为模板,根据设计好的引物进行PCR扩增,分别构建5个与HCM致病相关热点突变位点(MYH7-c.1987CT、TNNI3-c.370GC、MYH7-c.2155CT、TNNI3-c.433CG和PRKAG2-c.298GA)的野生型、纯合突变型和杂合突变型阳性质控品。以阳性质控品为模板,对5对引物和探针组合物的灵敏性、重复性和特异性进行验证。研究结果表明,对阳性质控品10倍梯度稀释(10~(-1)～10~(-7)),PCR扩增标准曲线R~20.996;批间和批内重复性验证其变异系数均小于2%;特异性验证显示,探针与模板匹配的荧光信号较强。以上结果表明,引物探针组合物的灵敏性、重复性和特异性较好。此外,以阳性HCM患者基因组为模板,对引物探针组合物进行回复性验证,其检测结果与毛细管电泳一致。综上所述,本研究利用Taqman-MGB实时荧光PCR检测技术,建立了经济、快速、灵敏的HCM热点突变位点检测方法。该方法的建立,有助于HCM临床分子诊断、治疗和愈后评估。     

Differential docking of high-affinity peptide ligands to type A and B cholecystokinin receptors demonstrated by photoaffinity labeling

Type A and B cholecystokinin (CCK) receptors are highly homologous members of the class-I family of G protein-coupled receptors that bind CCK with high affinity. However, they have divergent structural specificities, with the type A receptor requiring seven carboxyl-terminal residues including a sulfated tyrosine and the type B receptor requiring only the carboxyl-terminal tetrapeptide. The aim of this work was to utilize affinity labeling to determine spatial approximations with photolabile p-benzoyl-l-phenylalanine (Bpa) residues sited at each end of CCK as docked at the type B CCK receptor, contrasting this with analogous work using similar probes docked at the type A receptor. Both probes were fully efficacious, potent agonists that stimulated intracellular calcium in receptor-bearing CHO-CCKBR cells (EC(50) values: Bpa(24) probe, 41 +/- 9 pM; Bpa(33) probe, 15 +/- 3.3 pM). They bound specifically, with high affinity (K(i) values: Bpa(24) probe, 0.60 +/- 0.17 nM; Bpa(33) probe, 0.58 +/- 0.11 nM). Cyanogen bromide cleavage of the covalently labeled receptor suggested the first extracellular loop as the region of labeling by each probe, distinct from the type A CCK receptor regions labeled using the same probes (third loop and amino-terminal tail, respectively). This was confirmed by subsequent enzymatic and chemical cleavage of labeled wild-type and mutant receptors. Sequential cycles of Edman degradation of labeled receptor fragments identified the specific residues within loop one labeled by each probe (Bpa(24) probe labeled Phe(122); Bpa(33) probe labeled Thr(119)). This provides a direct demonstration of distinct modes of docking the same high-affinity ligand to highly homologous receptors.     

Differential localization of mRNAs of collagen types I and II in chick fibroblasts, chondrocytes, and corneal cells by in situ hybridization using cDNA probes

We have employed a highly specific in situ hybridization protocol that allows differential detection of mRNAs of collagen types I and II in paraffin sections from chick embryo tissues. All probes were cDNA restriction fragments encoding portions of the C-propeptide region of the pro alpha-chain, and some of the fragments also encoded the 3'-untranslated region of mRNAs of either type I or type II collagen. Smears of tendon fibroblasts and those of sternal chondrocytes from 17-d-old chick embryos as well as paraffin sections of 10-d-old whole embryos and of the cornea of 6.5-d-old embryos were hybridized with 3H-labeled probes for either type I or type II collagen mRNA. Autoradiographs revealed that the labeling was prominent in tendon fibroblasts with the type I collagen probe and in sternal chondrocytes with the type II collagen probe; that in the cartilage of sclera and limbs from 10-d-old embryos, the type I probe showed strong labeling of fibroblast sheets surrounding the cartilage and of a few chondrocytes in the cartilage, whereas the type II probe labeled chondrocytes intensely and only a few fibroblasts; and that in the cornea of 6.5-d-old embryos, the type I probe labeled the epithelial cells and fibroblasts in the stroma heavily, and the endothelial cells slightly, whereas the type II probe labeled almost exclusively the epithelial cells except for a slight labeling in the endothelial cells. These data indicate that embryonic tissues express these two collagen genes separately and/or simultaneously and offer new approaches to the study of the cellular regulation of extracellular matrix components.     

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples

A method based on three-DNA-component, sandwich hybridization has been designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into two vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these two reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was shown to be dependent on both the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 X 10(-6) pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.     

Fluorogenic probe for measuring high-mannose type glycan-specific endo-β-N-acetylglucosaminidase H activity

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the β-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.     

酶标DNA探针与Y染色体DNA的探测

 用辣根过氧化物酶标记DNA的技术,制备了酶标基因探针。研究了酶标过程和产物的电泳行为;用斑点杂交和southern印迹杂交探测了单链、双链DNA,灵敏度可达pg水平,以此酶标的Y染色体特异的DNA片段作探针,进行了DNA杂交的性别分析,证明该探针能清楚地区别两性基因组DNA,这对基因的研究和诊断有一定实用价值。     

Immunoelectron microscopic analysis of elongation of type 1 fimbriae in Escherichia coli.

Using 10- and 20-nm-diameter gold particles conjugated to an antifimbrial monoclonal antibody, we analyzed the location of assembly of newly formed subunits on growing type 1 fimbriae of Escherichia coli. Fimbriae were removed from an E. coli K-12-derived strain, CSH50, by blending. Blended cells were allowed to regenerate their fimbriae in growth medium for approximately 25 min, after which they were labeled with a 20-nm-gold-monoclonal antibody probe. Continued outgrowth of these labeled fimbriae was allowed for additional time intervals, after which they were labeled with a 10-nm-gold-monoclonal antibody probe. The resulting fimbriae, double labeled with 10- and 20-nm-diameter gold particles, were examined in an electron microscope. The pattern of labeling on individual fimbrial organelles indicated morphologically that newly synthesized subunits are added to a growing organelle at its base.     

The use of the fluorescence photobleaching recovery technique to study the self-assembly of tubulin

Fluorescently labeled microtubule-associated proteins or poly-L-lysine (13,000 MW) were prepared by reaction with fluorescein isothiocyanate. The labeled compounds were used as probes of the assembly of calf brain tubulin using fluorescence photobleaching recovery techniques which allow measurement of the diffusion coefficient and percentage mobility of the fluorescent probe. When unfractionated tubulin (defined as material containing tubulin and microtubule-associated proteins) was polymerized at room temperature or 37 degrees C, either probe could be incorporated into microtubules, since the observed diffusion coefficient (approximately 1.7 X 10(-8) cm2/s) was much slower than that for either probe free in solution. The microtubules formed in the presence of labeled microtubule-associated proteins were free to diffuse while those formed in the presence of labeled polylysine were partially immobilized. Thus the fluorescence photobleaching recovery technique can be used to measure crosslinking of microtubules as well as assembly or interactions with other structures. When unfractionated tubulin was incubated with labeled polylysine in the presence of Ca2+ at room temperature, the observed diffusion coefficient (approximately 5.1 X 10(-8) cm2/s) probably represents the formation of rings of tubulin. The effect of mild and vigorous shearing, of cholchicine, and of different Mg2+ concentrations on the properties of the system were examined.     

A polymerase chain reaction-based ribosomal DNA detection technique using a surface plasmon resonance detector for a red tide causing microalga, Alexandrium affine

A detection technique with a DNA probe was developed for the bloom‐forming alga Alexandrium affine harvested in Japan. The design of this probe was based on the sequence polymorphism within the 28S ribosomal DNA (rDNA) of this strain using the BIAcore? 2000 biosensor, which determines surface plasmon resonance. The specific DNA sequence in 28S rDNA for A. affine was determined by sequence data analysis, and a probe was designed for the detection of A. affine. A fragment of the 28S rDNA from A. affine was amplified by polymerase chain reaction and applied to the BIAcore? sensor system, and the target DNA was selectively recognized by species‐specific hybridization using two DNA probes: a fluorescein isothiocyanate (FITC)‐labeled probe and a biotin‐labeled DNA probe. Using FITC‐labeled anti‐immunogloblin G antibody, enhancement of the response for the target DNA can be detected directly as a resonant unit change. In this detection method, a difference within only 20 base pairs of the target could be detected, and specific detection of A. affine was achieved intraspecifically.     

近交系小鼠双色荧光正相杂交芯片技术体系的建立和优化

目的探讨采用单核苷酸多态性（SNP）检测方法-双色荧光正相杂交芯片技术对近交系小鼠遗传质量监测及相关影响因素。方法运用基于芯片的双色荧光正相杂交检测SNP技术,进行芯片杂交动力学研究,考察信号值（Cy3,Cy5）和ratio值（Cy5/Cy3）与PCR产物点样浓度、PCR产物长度和荧光标记探针长度之间的关系,研究PCR产物点样浓度、PCR产物长度和荧光标记探针长度对SNP分型的影响。结果采用正反标记实验后,Ratio值随着PCR产物点样浓度的增加呈稳定趋势;PCR双链产物长度对信号值影响比较大,点样时其长度不宜太长,最好不超过450 bp;随荧光标记探针长度的增加,基因分型能力明显下降,长度为15 bp最佳,长度超过20 bp时,已基本没有区分能力。结论PCR产物点样浓度、PCR产物长度和荧光标记探针长度是双色荧光正相杂交SNP分型系统的重要影响因素,采取适当的PCR产物点样浓度、PCR产物长度和荧光标记探针长度,并采用正反标记实验,可以取得稳定、准确的基因分型效果。为进一步进行近交系小鼠遗传质量监测的研究奠定基础。     

Use of a nitrotryptophan-containing peptide for photoaffinity labeling the pancreatic cholecystokinin receptor

We report the preparation and characterization of a new type of intrinsic photoaffinity labeling probe, on the basis of the incorporation of a photolabile nitrotryptophan into a biologically relevant domain of a peptide. The model system used was the pancreatic cholecystokinin (CCK) receptor, previously affinity labeled with a variety of probes. Those studies have suggested that an Mr = 85,000-95,000 protein is more likely to be labeled as the site of covalent attachment approaches the receptor-binding domain of this hormone. Indeed, CCK has a Trp in the center of its receptor-binding region, and replacement of that residue with 6-nitrotryptophan resulted in a photolabile probe which affinity labeled the same Mr = 85,000-95,000 pancreatic membrane protein. This probe, 125I-D-Tyr-Gly-[(Nle28,31,6-NO2-Trp30)CCK-26-33], was synthesized by solid-phase and solution techniques and characterized by mass spectrometry. Following oxidative iodination, it was purified on HPLC to 2000 Ci/mmol. Binding to pancreatic membranes was rapid, temperature dependent, reversible, saturable, and specific and was with high affinity (Kd = 3 nM). While its binding affinity was only 3-fold lower than that of native CCK-8, this probe was 70-fold less potent than native hormone in stimulating amylase secretion (EC50 = 1 nM) and equally efficacious to native hormone. Despite the slight decrease in affinity, this probe demonstrated a high relative efficiency of covalent labeling of the Mr = 85,000-95,000 protein. This confirms that the Mr = 85,000-95,000 protein represents the hormone-binding subunit of the CCK receptor and demonstrates the utility of this type of photoaffinity labeling probe.(ABSTRACT TRUNCATED AT 250 WORDS)