未修饰的纳米金结合等位特异性扩增来检测K-ras基因点突变

将等位基因特异性扩增的特异性与纳米金特殊的光学性质相结合, 发展了一种新的基因点突变检测方法. 以肿瘤中常见的K-ras癌基因第12位密码子作为点突变检测对象, 采用突变型引物对待测序列进行等位特异性扩增. 突变型样品扩增产物中大部分是双链DNA; 而野生型样品由于不能被顺利扩增, 产物中大部分是单链DNA. 以纳米金颗粒作为报告基团, 向两种不同基因型扩增产物中依次加入纳米金胶和盐溶液, 野生型基因扩增产物中的单链引物被吸附到纳米金颗粒表面, 使得纳米金在适宜浓度的盐溶液中不发生聚集; 突变型样品扩增产物中的双链DNA由于与纳米金颗粒间存在静电斥力而不能被吸附到纳米金颗粒表面, 纳米金在该浓度的盐溶液中发生聚集, 导致两种基因型的混合液在吸收光谱和颜色方面均存在显著差异, 从而实现了检测基因点突变的目的. 该检测方法直观、快速、简便, 实验成本低, 能够检测到pmol量级的样品, 为点突变检测提供了一种实用的新方法.     

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。     

电化学发光PCR定量检测H-ras癌基因点突变

提出了一种电化学发光PCR(ECL-PCR)分析方法,该法可用于定量检测基因点突变.其检测H-ras癌基因PCR扩增产物的灵敏度可达100fmol;线性范围为0.1-500pmol.用ECL-PCR分析法对膀胱癌组织中H-ras癌基因进行突变检测,只需要10μL样品,20min的孵育时间和30s的采集时间,得出20例膀胱癌样品中有7例存在点突变,通过标准曲线方程定量计算出突变样品的量.ECL-PCR分析方法在灵敏度、线性范围、分析时间等方面都优于传统的检测方法,是一种安全、快速、灵敏、定量检测基因点突变的分析方法.     

一种基于磁性纳米粒子PCR的高通量SNP分型方法

利用磁性纳米粒子PCR扩增(MNPs-PCR)和等位基因特异性双色荧光探针(Cy3, Cy5)杂交, 建立了一种单核苷酸多态性(SNP)分型的新方法. 应用该方法对9个样本MTHFR基因的C677T多态进行检测, 野生和突变型样本正错配信号比大于9.0, 杂合型正错配信号比接近1.0, 分型结果经测序验证. 此方法无须产物纯化、浓缩, 扫描分型结果快速、直观, 是一种操作简单、快速、高通量、高灵敏度的分型方法.     

结直肠癌组织中K-ras基因突变的毛细管电泳检测

通过对PCR扩增的76例结直肠癌组织及癌旁正常组织DNA基因组共152个样本纯化变性后,采用毛细管电泳-激光诱导荧光检测(CE-LIF)结合单链构象多态性(SSCP)分析方法检测了人结直肠癌组织及癌旁正常组织中K-ras基因第12/13位密码子突变。所检测的76例结直肠癌患者中有30例患者存在基因突变,并对异常片段进行测序验证,测序证实以碱基G→A点突变为主。结果表明所建立的CE-LIF技术结合SSCP分析检测K-ras基因突变的方法高效、快速、灵敏、准确,适合于临床上大样本结直肠癌中K-ras基因突变分析,对选择抗结直肠癌药物有一定的指导作用。     

毛细管电泳检测肺癌基因突变的方法学研究

建立了一种毛细管电泳快速高效检测聚合酶链反应(PCR)扩增产物以及限制性内切酶酶切产物的方法,使其更好地用于基因诊断.以聚环氧乙烷(poly(ethylene oxide),PEO)为筛分介质,用涂层的毛细管柱(37 cm×75 μm,有效长度27 cm)分离pUC19 DNA/MspⅠ(HpaⅡ) Marker标准DNA片段.考察了筛分介质的质量浓度、pH值、毛细管柱的温度和运行电压.在1×TBE (pH 8.2)电泳液、电压15 kV、温度15 ℃,于10 min内成功分离了Marker标准DNA片段.该方法快速、灵敏、准确,用于临床76例肺癌患者正常组织和肿瘤组织p53基因和ras基因点突变情况的检测,结果满意.     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

基于DNA聚合酶I的新型电化学传感器及其胰腺癌K-ras基因点突变检测

本文构建DNA聚合酶I的新型DNA电化学传感器,将捕获探针通过Au-S键固定于Au基底表面,与互补靶序列杂交至点突变前一个碱基,通过DNA聚合酶Ⅰ将dUTP-biotin连接在目标DNA的检测位点,再与avidin-HRP反应,而后测定在TMB溶液中的电化学特性. 结果表明,DNA电化学传感电极的检测电流值与K-ras突变型基因浓度（1.0×10^-15 ~ 1.0×10^-10 mol·L^-1）对数呈良好的线性关系,且灵敏度高,特异性较佳.     

基于核酸外切酶Ⅲ信号放大技术的荧光适配体传感器用于卡那霉素的检测

建立了一种基于核酸外切酶Ⅲ(Exo Ⅲ)和碳纳米颗粒(CNPs)的信号放大体系用于卡那霉素(KAN)检测的新方法。合成了水溶性的CNPs,并设计合成了不同序列的DNA,具体包括:卡那霉素适配体(Apt),羧基荧光素标记的信号DNA探针(FAM-DNA)和互补链cDNA。当体系中不存在KAN时,Apt与cDNA可以杂交形成双链DNA,体系中FAM-DNA处于单链状态,Exo Ⅲ不能水解单链DNA;此时,体系中加入CNPs,单链FAM-DNA被CNPs吸附,荧光发生淬灭;在KAN存在下,Apt与其靶标KAN特异性结合,此时FAM-DNA与cDNA杂交形成双链DNA,由于CNPs对双链DNA吸附较弱,DNA探针的荧光不发生淬灭。ExoⅢ可以特异性的从3’-端对FAM-DNA降解,释放FAM荧光团和cDNA,该体系通过"降解-杂交"循环,最终释放出大量的FAM荧光团。由于CNPs对FAM具有较低的亲和力,释放出的FAM不能吸附在CNPs表面,FAM荧光不会发生淬灭,实现荧光信号放大扩增作用。方法线性范围为50～100 nmol/L,检测限为2.5 nmol/L。该方法可用于实际样品牛奶中卡那霉素的检测。     

基于DNA双链取代策略免标记检测铅离子的研究

建立了一种基于DNA双链取代策略和SYBR GreenⅠ(SG)作为荧光染料插入剂进行免标记铅离子检测的荧光传感方法。SG作为一种染料分子,与单链DNA作用产生的荧光强度很弱,但可以插入双链DNA,使SG荧光强度明显增强。检测时铅离子适配体首先与其部分互补单链DNA杂交形成稳定的双链DNA结构,当溶液中存在铅离子时,铅离子与其适配体特异性结合,双链DNA的数量减少,加入SG可实现铅离子的免标记定量检测。此方法具有灵敏度高、特异性强、简便快速等优点。最低检测浓度为2 nmol/L,检出限(S/N=3)为1.6 nmol/L,实际样品检测结果良好。     

A Novel Real-time Fluorescence Mutant-allele-specific Amplification Method for Rapid Single Nucleotide Polymorphism Analysis

Current methods for single nucleotide polymorphism (SNP) analysis are time-consuming and complicated. We aimed at development of one-step real-time fluorescence mutant-allele-specific amplification (MASA) method for rapid SNP analysis. The method is a marriage of two technologies: MASA primers for target DNA and a double-stranded DNA-selective fluorescent dye, SYBR Green I. Genotypes are separated according to the different threshold cycles of the wild-type and mutant primers. K-rar oncogene was used as a target to validate the feasibility of the method. The experimental results showed that the different genotypes can be clearly discriminated by the assay. The real-time fluorescence MASA method will have an enormous potential for fast and reliable SNP analysis due to its simplicity and low cost.     

Highly sensitive ECL-PCR method for detection of K-ras point mutation

A highly sensitive electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for K-ras point mutation detection is developed. Briefly, K-ras oncogene was amplified by a Ru(bpy)32 (TBR)-labeled forward and a biotin-labeled reverse primer, and followed by digestion with MvaI restriction enzyme, which only cut the wild-type amplicon containing its cutting site. The digested product was then adsorbed to the streptavidin-coated microbead through the biotin label and detected by ECL assay. The experiment results showed that the different genotypes can be clearly discriminated by ECL-PCR method. It is useful in point mutation detection, due to its sensitivity, safety, and simplicity.     

Approaching real-time molecular diagnostics: single-pair fluorescence resonance energy transfer (spFRET) detection for the analysis of low abundant point mutations in K-ras oncogenes

The aim of this study was to develop new strategies for analyzing molecular signatures of disease states approaching real-time using single pair fluorescence resonance energy transfer (spFRET) to rapidly detect point mutations in unamplified genomic DNA. In addition, the detection process was required to discriminate between normal and mutant (minority) DNAs in heterogeneous populations. The discrimination was carried out using allele-specific primers, which flanked the point mutation in the target gene and were ligated using a thermostable ligase enzyme only when the genomic DNA carried this mutation. The allele-specific primers also carried complementary stem structures with end-labels (donor/acceptor fluorescent dyes, Cy5/Cy5.5, respectively), which formed a molecular beacon following ligation. We coupled ligase detection reaction (LDR) with spFRET to identify a single base mutation in codon 12 of a K-ras oncogene that has high diagnostic value for colorectal cancers. A simple diode laser-based fluorescence system capable of interrogating single fluorescent molecules undergoing FRET was used to detect photon bursts generated from the molecular beacon probes formed upon ligation. LDR-spFRET provided the necessary specificity and sensitivity to detect single-point mutations in as little as 600 copies of human genomic DNA directly without PCR at a level of 1 mutant per 1000 wild type sequences using 20 LDR thermal cycles. We also demonstrate the ability to rapidly discriminate single base differences in the K-ras gene in less than 5 min at a frequency of 1 mutant DNA per 10 normals using only a single LDR thermal cycle of genomic DNA (600 copies). Real-time LDR-spFRET detection of point mutations in the K-ras gene was accomplished in PMMA microfluidic devices using sheath flows.     

Analysis of polymerase chain reaction-product by capillary electrophoresis with laser-induced fluorescence detection and its application to the diagnosis of medium-chain acyl-coenzyme A dehydrogenase deficiency

Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) has been developed to detect polymerase chain reaction (PCR) amplified samples. LIF detection was performed using Thiazole Orange as the fluorescent intercalating dye. This method was ca. 100X as sensitive as that with UV detection. The highly sensitive CGE-LIF was applied to the detection of the most prevalent mutation (lysine³²⁹- to-glutamic acid substitution) in medium-chain acyl-coenzyme A dehydrogenase (MCAD) deficiency. The disorder, which shows an autosomal recessive inheritance, is known to be highly prevalent among Caucasian population and often mimics as Reye-like syndrome or sudden infant death.

A DNA fragment containing the mutation site was PCR-amplified with two sets of allele specific oligonucleotide primers, followed by CGE-LIF. The mutant allele produced a 175-base pairs DNA fragment, which the normal allele generated a 202-base pairs DNA fragment. CGE-LIF clearly distinguished these PCR products, facilitating rapid diagnosis of MCAD deficiency.     

Allele-specific amplification and electrochemiluminescence method for single nucleotide polymorphism analysis

A new approach combined the specificity of allele-specific amplification (ASA) with the sensitivity of electrochemilu- minescence (ECL) assay for single nucleotide polymorphism (SNP) analysis was proposed. Briefly, target gene was amplified by a biotin-labeled allele-specific forward primer and a Ru(bpy)32 (TBR)-labeled universal reverse primer. Then, the amplicon was captured onto streptavidin-coated paramagnetic beads through biotin label, and detected by measuring the ECL signal of TBR label. Different genotypes were distinguished according to the ECL values of the amplicons by different genotypic primers. K-ras oncogene was used as a target to validate the feasibility of the method. The experiment results show that the different genotypes can be clearly distinguished by ASA–ECL assay. The method is useful in SNP analysis due to its sensitivity, safety, and simplicity.     

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.     

A sensitive fluorescence anisotropy method for point mutation detection by using core-shell fluorescent nanoparticles and high-fidelity DNA ligase

The present study reports a proof-of-principle for a sensitive genotyping assay approach that can detect single nucleotide polymorphisms (SNPs) based on fluorescence anisotropy measurements through a core-shell fluorescent nanoparticles assembly and ligase reaction. By incorporating the core-shell fluorescent nanoparticles into fluorescence anisotropy measurements, this assay provided a convenient and sensitive detection assay that enabled straightforward single-base discrimination without the need of complicated operational steps. The assay was implemented via two steps: first, the hybridization reaction that allowed two nanoparticle-tagged probes to hybridize with the target DNA strand and the ligase reaction that generated the ligation between perfectly matched probes while no ligation occurred between mismatched ones were implemented synchronously in the same solution. Then, a thermal treatment at a relatively high temperature discriminated the ligation of probes. When the reaction mixture was heated to denature the duplex formed, the fluorescence anisotropy value of the perfect-match solution does not revert to the initial value, while that of the mismatch again comes back as the assembled fluorescent nanoparticles dispart. The present approach has been demonstrated with the discrimination of a single base mutation in codon 12 of a K-ras oncogene that is of significant value for colorectal cancers diagnosis, and the wild type and mutant type were successfully scored. Due to its ease of operation and high sensitivity, it was expected that the proposed detection approach might hold great promise in practical clinical diagnosis.