毛细管电泳检测肺癌及癌旁正常组织蛋白质混合物差异

以建立的毛细管电泳(CE)-激光诱导荧光(LIF)检测蛋白质的方法对提取肺癌及癌旁正常组织蛋白质混合物(变 性/活性)差异进行检测. 采用异硫氰酸荧光素(FITC)为衍生剂, 电泳缓冲液为1×TBE (TBE为Tris-硼酸-EDTA, 变性电泳pH 10.0, 活性电泳为pH 8.3且含有2 mg/L考马斯亮蓝), 分离电压15 kV, 柱温15 ℃, 电动进样(10 kV×10 s), 激发波长/发射波长＝488/520 nm检测时, 肺癌及癌旁正常组织蛋白质混合物样品得到较好分离且有明显差异. 与目前常用蛋白分析方法: 变性SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)以及活性蓝绿温和胶电泳(BN-PAGE)进行比较. BN-PAGE结果显示肺癌组织相比正常组织有较明显蛋白种类差异|SDS-PAGE结果表明一些蛋白质表达量差异也是肺癌及癌旁正常组织的显著差别, 且主要集中在20～116 kDa. CE-LIF检测结果与PAGE结果大致相同, 且CE-LIF检测蛋白质的灵敏度高于PAGE, 能更准确反映肺癌及癌旁正常组织的蛋白质差异. 结论是CE-LIF可用于蛋白质差异检测, 时间短, 效果较好, 对活性蛋白质进行分析体现了其优点: 可提供较强的动力——电压, 及强动力下良好的温度稳定性.     

毛细管电泳法检测癌基因C-myc胃癌中基因点突变

癌基因C-myc激活和突变在胃癌形成过程中起着重要作用。通过毛细管电泳(CE)方法检测50例胃癌患者中C-myc基因突变,建立一种准确、快速诊断早期胃癌的方法。本实验采用PCR扩增胃癌及癌旁正常组织中C-myc基因第二外显子易发突变的部位基因序列,扩增样品分别经96℃变性和EcoRⅤ酶切处理,以PAGE-SSCP,CE-SSCP,CE-RFLP分别对其突变情况进行检测。优化的CE检测条件:筛分介质PEO浓度3.0%,pH 8.2,电压15 kV,温度15℃;荧光检测:λex=488 nm,λem=520 nm。检测结果:C-myc基因总突变率为20.0%(10/50)。测序分析结果显示C-myc基因第二外显子第53密码子存在点突变,碱基A变为碱基T(GAT→GTT),碱基的改变使氨基酸由亮氨酸替代为谷氨酰胺。本研究数据证实C-myc基因突变与胃癌的形成紧密相关,CE检测C-myc突变基因可作为胃癌早期诊断的简便可靠的方法。     

基于热阻梯度加热系统的温度梯度芯片毛细管电泳用于DNA突变检测

建立了一种简单、可靠的空间温度梯度芯片毛细管电泳DNA突变分析系统, 制作了热阻呈梯度均匀变化的硅橡胶(PDMS)基片, 利用其热阻变化对热传导的影响, 在基片表面形成稳定的空间温度梯度. 通过改变PDMS基片的厚度差, 可得到范围不同的温度梯度, 且形成的温度梯度在6 h内保持稳定. 利用该温度梯度加热装置对玻璃微流控芯片进行加热, 在10 ℃温度梯度范围内对209 bp的DNA突变标准样品进行分离检测, 单次样品分析时间为8.3 min, 并成功用于3例大肠癌患者石蜡组织切片中K-ras基因突变的检测.     

毛细管电泳-单链构象多态性分析检测K-ras基因突变

K ras癌基因的点突变在结直肠癌的发生起重要作用。以异丙醇为聚合反应链转移剂,水相法合成 特性粘度为0.70×10-3m3/kg,分子量为6.5×104的低粘度短链线性聚丙烯酰胺。以6%线性聚丙烯酰胺为 筛分介质,分离温度27℃,分离电压9kV为电泳条件,建立了检测K ras基因突变的毛细管电泳 单链构象多 态性方法。利用该方法检测36例结直肠癌患者肿瘤组织,发现12例K ras基因突变。结果表明:该方法具有 快速、高灵敏的优点,为大规模进行结直肠肿瘤的早期诊断提供了可靠方法。     

毛细管电泳法检测胃癌APC基因第十一外显子的杂合缺失

采用聚合酶链反应(PCR)扩增了胃癌及癌旁正常组织中APC基因易发生杂合缺失的第十一外显子的部分碱基序列,扩增样品分别经96℃变性和Rsa Ⅰ酶切处理,以毛细管电泳(CE)-单链构象多态性(SSCP)、CE-限制性片段长度多态性(RFLP)、聚丙烯酰胺凝胶电泳(PAGE)-SSCP对其杂合缺失情况进行检测.PAGE凝胶...     

实时荧光等位基因特异性扩增法快速检测K-ras癌基因点突变

将荧光定量PCR技术与等位基因特异性扩增(Allele specific amplification, ASA)方法相结合, 发展了一种可以快速检测基因点突变的实时荧光等位基因特异性扩增(Real-time ASA)方法. 将该法用于检测K-ras癌基因第12位密码子发生的点突变, 分别采用针对其不同点突变方式(GAT, GTT, CGT)设计的突变型引物对待测样品进行ASA, 只有突变型样品能被顺利扩增出双链DNA产物, 该产物才能与双链DNA染料SYBR Green Ⅰ结合, 发出荧光信号从而被检测到. 用该法检测31例结肠癌组织中的K-ras癌基因点突变, 其中有15例样品检出为突变型. Real-time ASA法可检测到样品中含量为1/1000的突变型基因, 具有灵敏、快速、简便、安全、高通量和低成本等优点, 可望用于大量临床样本的点突变筛查.     

多态性及基因测序分析方法研究苯丙酮尿症患者的基因突变

应用聚合酶链反应-单链构象多态性分析及基因测序的方法,分析35例苯丙酮尿症患者PAH基因第6和7外显子以及其两侧部分内含子序列,进行了突变的筛查和确定.研究吉林地区苯丙酮尿症人群中苯丙氨酸羟化酶(Phenylalanine hydroxylase,PAH)基因突变特征.结果显示:(1)在70个PAH等位基因中共检测出9...     

人原发性肝癌中N-ras基因的表达

对9例人原发性肝癌,1例癌旁组织,1例正常肝的poly(A)~ RNA进行了分析。用各种癌基因探针作分子杂交,发现在6例原发性肝癌中,有二条增强表达的区带:2.2kb和5.6kb。肝癌组织较正常肝的mRNA在2.2kb处有明显的增强。提示人N-ras基因的转录产物明显增高。癌旁,正常肝中N-ras基因的专一的mRNA很弱或低于检测水平。由于癌的发生是通过癌基因产物发生作用,因此N-ras基因在多数的人原发性肝癌中的表达明显增强,提示了N-ras基因是人原发性肝癌的重要转化基因之一。     

MiR-335在口腔鳞状细胞癌中的表达变化

目的探明miR-335在口腔鳞状细胞癌组织中的表达异常情况。方法采用qRT-PCR分别检测25例人口腔鳞状癌细胞及相应邻近正常黏膜组织中miR-335的表达情况,对miR-335在口腔鳞状细胞癌及相应邻近正常黏膜组织中的表达情况进行比较。结果 miR-335在口腔鳞状细胞癌中的表达显著低于其癌旁正常黏膜组织(P0.05)。结论 miR-335在舌鳞癌中呈低表达,有可能在口腔鳞状细胞癌的发生、发展起重要作用。     

电化学发光-聚合酶链式反应方法检测胰腺癌中K-ras基因点突变

发展了一种可用于快速检测胰腺癌中K-ras癌基因点突变的电化学发光-聚合酶链式反应(ECL-PCR)分析方法。该法采用三联吡啶钌标记的上游引物和生物素标记的下游引物对目的片段进行PCR扩增;再采用限制性内切酶MvaI对扩增产物进行酶切。由于野生型样品和突变型样品间存在酶切位点的变化,其中只有野生型样品能被切断;通过生物素与链霉亲和素包被的磁珠连接,将生物素标记的DNA片段收集到检测池中,进行电化学发光检测。采用该法对13例胰腺癌组织中的K-ras癌基因第12位密码子进行点突变分析,只需要10μL样品、20min孵育时间和30s采集时间,就可得出其中有12例存在点突变,点突变率为92.3%。本方法操作简便、安全、快速、灵敏,可用于检测任何一种导致限制性内切酶位点改变的基因点突变。     

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.     

Applications of homemade kit in mutation detection of genes

With the accomplishment of Human Genome Project (HGP), single nucleotide polymorphism (SNP) and mutation detection in human genome are becom-ing a new researching focus. These researches can help us to understand the phenotype diversity of indi-vidual, disease susceptibility and drug resistance of different colonies. Traditional method used for muta-tion detection is slab gel electrophoresis, which re-mains labor-intensive and time-consuming because of the requirement of radioactivity or te…     

Detection of K-ras exon 1 mutations by constant denaturant capillary electrophoresis

Among various mutation detection methods, constant denaturant capillary electrophoresis (CDCE) is one of the most common techniques for rapid identification of known or unknown mutations. In this report, a CDCE analysis method with homemade linear polyacrylamide (LPA) kit was developed on ABI 310 genetic analyzer, the effect and relationship of various denaturing factors in CDCE analysis were investigated and K-ras gene mutations of 31 coloerctal cancer patients were detected. Results indicate that, with the increase of chemical danaturant concentration, the optimum temperature was lowered, and when the concentration of urea (formamide) was higher than 7 M (40%), the homoduplex and heteroduplex of mutant samples were separated with difficulty. Detection results of K-ras gene in colorectal samples indicated that mutations were present in eight (26%) of 31 patients; most mutations were localized in codon 12, which is thought to be a critical step and plays an important role in human colorectal carcinogenesisas.     

Identification of the commonest cystic fibrosis transmembrane regulator gene DeltaF508 mutation: evaluation of PCR--single-strand conformational polymorphism and polyacrylamide gel electrophoresis

In the present study we investigated whether single-strand conformational polymorphism (SSCP) and polyacrylamide gel electrophoresis (PAGE) could be used for the identification of the CFTR DeltaF508 gene mutation, which is commonest in the Greek population. Using DNA from patients carrying this mutation, the appropriate 98 bp region of the CFTR gene was amplified by PCR and the reaction products were analysed by non-radioactive SSCP-electrophoresis using silver staining for band visualization and non-denaturating PAGE to confirm the results. SSCP electrophoretic analysis has been optimized for several parameters in order to achieve the best resolution. Single-strand DNA fragments gave a reproducible pattern of bands, characteristic for the particular mutation. Comparison of the obtain patterns with control samples allowed the detection of the DeltaF508 mutation in the patients studied by SSCP assay and these results were confirmed by the independent method of PAGE. Although SSCP and PAGE can be used for detection of this mutation, PAGE resulted in more distinct patterns than SSCP. It is, therefore, proposed that PAGE can be reliably used for the detection and identification of such a mutation in patients provided that suitable controls are available. The applicability of PAGE to identification of the mutation in carriers, particularly useful for population screening, is also discussed.     

DNA Extraction from Formalin-fixed and Paraffin-embedded Tissues by Triton X-100 for Effective Amplification of EGFR Gene by Polymerase Chain Reaction

For first-line non-small-cell lung cancer(NSCLC) therapy, detecting mutation status of the epidermal growth factor receptor(EGFR) geneconstitutes a prudent test to identify patients who are most likely to benefit from EGFR-tyrosine kinase inhibitor(TKI) therapy. Now, the material for detecting EGFR gene mutation status mainly comes from formalin-fixed and paraffin-embedded(FFPE) tissues. DNA extraction from FFPE and the amplification of EGFR gene by polymerase chain reaction(PCR) are two key steps for detecting EGFR gene mutation. We showed a simple method of DNA extraction from FFPE tissues for the effective amplification of EGFR gene. Extracting DNA from the FFPE tissues of NSCLC patients with 1% Triton X-100(pH=10.0) was performed by heating at 95℃ for 30 min. Meanwhile, a commercial kit was used to extract DNA from the same FFPE tissues of NSCLC patients for comparison. DNA extracted products were used as template for amplifying the exons 18, 19, 20 and 21 of EGFR by PCR for different amplified fragments. Results show that DNA fragment size extracted from FFPE tissues with 1% Triton X was about 250-500 base pairs(bp). However,DNA fragment size extracted from FFPE tissues via commercial kit was about from several hundreds to several thousands bp. The DNA yield extracted from FFPE tissues with 1% Triton X was larger than that via commercial kit. For about 500 bp fragment, four exons of EGFR could not be amplified more efficiently from extracted DNA with 1% Triton X than with commercial kit. However, for about 200 bp fragment. This simple and non-laborious protocol could successfully be used to extract DNA from FFPE tissue for the amplification of EGFR gene by PCR, further screening of EGFR gene mutation and facilitating the molecular analysis of a large number of FFPE tissues from NSCLC patients.     

Application of cycling gradient capillary electrophoresis to detection of APC, K-ras, and DCC point mutations in patients with sporadic colorectal tumors

A previously introduced technique of cycling gradient capillary electrophoresis (CGCE) was applied to monitoring of molecular changes during adenoma-carcinoma transition in progression of sporadic colorectal cancer. The purpose of this work was optimization of separation parameters for selected mutation regions in tumor suppressor genes involved in the early stages of colorectal carcinogenesis, followed by scanning for these mutations in clinical tissue samples from patients with adenomatous polyps and early carcinomas. A total of 47 colorectal tumors in various stages of progression were examined. Main emphasis was given to evaluation of mutation detection sensitivity and specificity required for effective early disease detection. A total of 7 different somatic mutations was identified among 32 K-ras mutant samples, 1 inherited mutation and 5 somatic mutations were identified among 15 adenomatous polyposis coli (APC) mutated samples. None of the two previously reported "deleted in colorectal carcinomas" (DCC) mutations was found in any of the clinical samples. In addition to simple optimization of running conditions, CGCE has demonstrated sensitivity and selectivity allowing detecting small mutant fractions as well as combination of multiple mutants within a single target sequence.     

Rapid identification of pathogenic bacteria by capillary electrophoretic analysis of rRNA genes

Molecular diagnosis is playing an increasingly important role in the rapid detection and identification of pathogenic organisms in clinical samples. The genetic variation of ribosomal genes in bacteria offers an alternative to culturing for the detection and identification of these organisms. Here 16S rRNA and 16S-23S rRNA spacer region genes were chosen as the amplified targets for single-strand conformation polymorphism (SSCP) and restriction fragment length polymorphism (RFLP) capillary electrophoresis analysis and bacterial identification. The multiple fluorescence based SSCP method for the 16S rRNA gene and the RFLP method for the 16S-23S rRNA spacer region gene were developed and applied to the identification of pathogenic bacteria in clinical samples, in which home-made short-chained linear polyacrylamide (LPA) was used as a sieving matrix; a higher sieving capability and shorter analysis time were achieved than with a commercial sieving matrix because of the simplified template preparation procedure. A set of 270 pathogenic bacteria representing 34 species in 14 genera were analyzed, and a total of 34 unique SSCP patterns representing 34 different pathogenic bacterial species were determined. Based on the use of machine code to represent peak patterns developed in this paper, the identification of bacterial species becomes much easier.     

Genotyping of K-ras codons 12 and 13 mutations in colorectal cancer by capillary electrophoresis

Point mutations of the K-ras gene located in codons 12 and 13 cause poor responses to the anti-epidermal growth factor receptor (anti-EGFR) therapy of colorectal cancer (CRC) patients. Besides, mutations of K-ras gene have also been proven to play an important role in human tumor progression. We established a simple and effective capillary electrophoresis (CE) method for simultaneous point mutation detection in codons 12 and 13 of K-ras gene. We combined one universal fluorescence-based nonhuman-sequence primer and two fragment-oriented primers in one tube, and performed this two-in-one polymerase chain reaction (PCR). PCR fragments included wild type and seven point mutations at codons 12 and 13 of K-ras gene. The amplicons were analyzed by single-strand conformation polymorphism (SSCP)-CE method. The CE analysis was performed by using a 1× Tris–borate–EDTA (TBE) buffer containing 1.5% (w/v) hydroxyethylcellulose (HEC) (MW 250 000) under reverse polarity with 15 °C and 30 °C. Ninety colorectal cancer patients were blindly genotyped using this developed method. The results showed good agreement with those of DNA sequencing method. The SSCP-CE was feasible for mutation screening of K-ras gene in populations.