一种基于磁性颗粒和通用连接子扩增技术的单核苷酸多态性分型方法

提出了一种应用磁性颗粒和通用连接子扩增技术(Linker-PCR)的多位点单核苷酸多态性(SNP)分型方法. 该方法首先通过酶切将样本基因组DNA打断, 然后将通用连接子通过T4 DNA连接酶与各个酶切片段连接, 利用生物素标记的通用引物将样本进行全基因组扩增. 扩增后, 将生物素标记的Linker-PCR扩增产物固定到亲合素修饰的磁性颗粒表面, 通过与双色荧光标记的等位基因特异性探针杂交, 对待测位点进行分型. 利用该方法, 我们对10个样本MTHFR基因上的2个SNP位点进行了分型, 分型结果准确、正错配信号比大于3. 由于利用Linker-PCR技术来实现对靶序列的全基因组扩增, 该方法非常适用于大量样本的多基因多位点的SNP分型研究.     

基于荧光小分子2-氨基-7-甲基-1,8-萘啶对C碱基的识别检测DNA双链中的C/C错配

单碱基错配是单核苷酸多态性(SNPs)的一种,是导致突变的DNA损伤类型之一.单碱基错配的检测对于从分子水平上阐明多种疾病形成的原因,实现基因水平的治疗都是至关重要的前提条件.发展具有高灵敏度、高选择性的单碱基错配检测方法势在必行.常用的单碱基错配检测方法包括凝胶电泳、荧光检测、SPR和质谱检测等.本文采用2-氨基-7-甲基-1,8-萘啶(AMND)作为荧光探针,AMND能嵌入双链DNA(ds-DNA)中的错配位点,并通过氢键识别错配碱基,这一结合过程伴随探针小分子的荧光淬灭,通过检测荧光淬灭现象实现单碱基错配及错配碱基类型的识别,建立了SNPs荧光分型方法.     

基于核酸外切酶Ⅲ信号放大的比率型荧光传感器检测致病菌基因

食源性致病菌严重威胁着公众健康。本研究基于荧光共振能量转移原理,以Cy3和Cy5作为荧光供体和受体基团,利用核酸外切酶Ⅲ(ExoⅢ)增强检测信号,构建了比率型荧光传感器,用于高灵敏度检测致病菌基因。分别标记有Cy3的R1-DNA和标记Cy5的R2-DNA形成双链R1/R2,在Cy3的激发光波长激发下,由于发生荧光共振能量转移,Cy3的荧光被猝灭而产生Cy5的荧光信号。致病菌靶基因(大肠杆菌Lac Z基因)的存在可将R1/R2双链解旋,使得Cy3远离Cy5,导致Cy3的荧光恢复而Cy5的荧光信号降低。在ExoⅢ作用下,Cy3与Cy5的信号变化进一步增大。在优化的实验条件下,荧光信号变化与靶基因在10~2000 pmol/L浓度范围内呈现良好的线性关系,检出限为5.29 pmol/L。所采用的比率型信号检测策略极大地降低了假阴性、假阳性检测结果的产生,增强了检测特异性。     

实时荧光聚合酶链式反应偶联高特异性核酸侵入反应检测单核苷酸多态性

建立了实时荧光聚合酶链式反应( PCR)偶联高特性核酸侵入反应检测单核苷酸多态性( SNP)的方法。优化了体系中flap核酸内切酶1(FEN1酶)和野生型检测探针等用量,确定了最佳反应条件,即FEN1酶用量为1.5 U,野生型检测探针用量为0.125μmol/L,0.5μmol/L Invader突变型检测探针,各0.25μmol/L通用野生型( VIC)和突变型( FAM)荧光共振转移发卡探针,显著降低了野生型样本和突变型样本背景信号,避免了背景信号对检测结果分型的干扰。采用本方法对编码乙醛脱氢酶2( ALDH2)基因ALDH2*2位点21例样本、细胞色素P4502C19基因CYP2C19*2和CYP2C19*3位点各19例样本进行分型检测,结果表明, AL-DH2*2位点GG纯合10例,GA杂合8例,AA纯合3例;CYP2C19*2位点GG纯合9例,GA杂合8例,AA纯合2例;CYP2C19*3位点GG纯合18例,GA杂合1例。使用焦磷酸测序进行验证,两种方法检测结果一致。本方法特异性好、操作简便、耗时短、成本低,可实现对SNP单管闭管无污染的分型检测。     

基于催化发夹组装和荧光共振能量转移的生物传感器检测EV71病毒

以肠道病毒71型(EV71)为检测对象，建立了基于催化发夹组装(CHA)和荧光共振能量转移(FRET)的生物传感器。设计了检测EV71 VP1基因的CHA信号放大策略，该体系由一对发夹探针(H1、 H2)构成，H1和H2分别标记了荧光基团Cy3和Cy5。当体系中有VP1基因时，可引发H1和H2催化自组装反应，从而使得Cy3和Cy5相互靠近并发生FRET,导致Cy3荧光信号降低，Cy5信号增强；当体系中没有VP1基因时，H1和H2则稳定存在于反应体系中，不发生FRET,只检测到Cy3的荧光信号。优化了缓冲液浓度、 H1与H2的比例、反应温度和反应时间。在最优条件下，所构建的传感器的Cy5与Cy3的荧光强度比值与EV71 VP1基因浓度在0.5～20 nmol/L范围内呈良好的线性关系，检出限为73 pmol/L(3σ)。咽拭子样品的加标回收率为99.6%～103.1%,相对标准偏差在0.3%～1.7%之间。本方法具有较好的特异性及抗干扰能力，在EV71监测和手足口病早期诊断方面具有良好的应用前景。     

基于聚多巴胺纳米粒子的荧光共振能量转移法检测microRNA

基于聚多巴胺纳米粒子(PDA NPs)对Cy5标记单链DNA(Cy5-ssDNA)探针的荧光猝灭效应以及脱氧核糖核酸酶Ⅰ(DNaseⅠ)选择性切割DNA/RNA杂合结构中单链DNA的特性,建立了一种用于微小核糖核酸(miRNA)检测的新型恒温信号放大方法.在优化的实验条件下,体系的相对荧光强度(FR)与miR-21浓度的对数值成正比;对miR-21检测的线性范围为10 pmol/L~100 nmol/L,检出限达7 pmol/L.血清加标实验结果表明,该方法可用于生理环境下miR-21的检测.     

八元瓜环对氯化矢车菊素溶解性、稳定性及抗氧化性的影响

采用核磁共振氢谱(¹H NMR)、 红外光谱及紫外吸收光谱等方法考察了八元瓜环(Q[8])对氯化矢车菊素(Cy)的包结作用. 结果表明, 在pH=0.8的盐酸介质中, Q[8]可与Cy形成摩尔比为1:1的主客体配合物, 紫外吸收光谱法测得的主-客体结合常数为1.51×10⁶ . 相溶解度研究结果表明, Q[8]能使饱和Cy溶液的溶解度增大, 当Q[8]浓度为100 μmol/L时, 可使Cy的溶解度增大12.21倍. 紫外吸收光谱随时间变化结果表明, 在较为稳定存在形态及相同实验条件下, Q[8]/Cy溶液比Cy溶液的稳定性提高了2.58倍. 抗氧化性实验结果表明, Q[8]/Cy包合物和Cy均表现出较好的抗氧化活性.     

两种菁类染料复合敏化TiO2纳米晶电极的光电化学研究

应用光电化学方法研究了两种菁类染料Cy3和Cy5复合敏化TiO2纳米晶电极的光电化学行为. 结合两种染料的紫外-可见光谱和循环伏安曲线, 确定了Cy3和Cy5的电子基态和激发态能级位置. 结果表明两种染料的激发态能级位置能与TiO2纳米粒子导带边位置相匹配, 复合敏化可以显著提高TiO2纳米晶的光电流, 使TiO2纳米晶电极吸收波长由紫外光区红移至可见光区和近红外区. 复合敏化降低了染料Cy3在电极吸附时的聚集程度, 使其单色光的转换效率(IPCE)提高了169%, 复合敏化电极总的光电转换效率η为2.09%, 分别是Cy3和Cy5单独敏化时光电转换效率的2.069和1.229倍.     

两种菁类染料复合敏化TiO_2纳米晶电极的光电化学研究

应用光电化学方法研究了两种菁类染料Cy3和Cy5复合敏化TiO2纳米晶电极的光电化学行为.结合两种染料的紫外-可见光谱和循环伏安曲线,确定了Cy3和Cy5的电子基态和激发态能级位置.结果表明两种染料的激发态能级位置能与TiO2纳米粒子导带边位置相匹配,复合敏化可以显著提高TiO2纳米晶的光电流,使TiO2纳米晶电极吸收波长由紫外光区红移至可见光区和近红外区.复合敏化降低了染料Cy3 在电极吸附时的聚集程度,使其单色光的转换效率(IPCE)提高了169%,复合敏化电极总的光电转换效率η为2.09%,分别是Cy3和Cy5单独敏化时光电转换效率的2.069 和1.229倍.     

基于双色荧光传感器的癌细胞成像及microRNA定量检测

癌细胞中microRNA(miRNA)的灵敏成像对于疾病的诊断治疗具有重要意义, 其中miRNA-21通常在多种癌细胞中异常表达. 本文将DNA功能化的金纳米颗粒与发射波长分离的荧光染料FAM和Cy5.5修饰的DNA通过含有光控基团PC-linker的DNA4作为桥梁进行自组装, 构建了纳米传感器GDC. 将302 nm紫外光作为启动开关, 用其照射该体系时, Cy5.5修饰的DNA3被释放, 其荧光强度可作为内参比信号, 用于标定进入细胞的组装体含量; 细胞中miRNA-21作为催化分子, 与外加燃料Fuel DNA共同作用下可实现催化放大, FAM修饰的DNA2被释放且被猝灭的荧光信号得以恢复, 并作为检测信号. 通过2种荧光信号强度(FL)的检测及FL_FAM/FL_Cy5.5比值的计算, 达到定量分析细胞中miRNA含量的目的. 该体系可扣除因细胞内组装体含量不同造成的背景信号误差, 不仅能显著提高检测准确度, 还因存在催化循环而大大降低了检出限, 比传统方法至少降低了3个数量级. 该传感器的检出限为23.1 pmol/L, 通过定量计算得出HeLa细胞中miRNA的含量为0.0236 nmol/L.     

Multiple genotyping based on multiplex PCR and microarray

The genetic variability has obtained more and more attention in the process of diagnosis and treatment of tumors.Herein,we have described a multiple genotyping method based on magnetic enrichmentmultiplex PCR (MEM-PCR) and microarray technology.Monodisperse magnetic beads were fabricated and modified with streptavidin.Four loci on two genes (M235T and A-6G loci on AGT gene,A1298C and C677T loci on MTHFR gene) were selected to study single nucleotide polymorphisms (SNP).Target sequences of these SNP loci were amplified using Cy3-labeled primers through multiplex PCR in one tube after the templates were enriched and purified by functional magnetic beads (MB).Four pairs of NH₂-labeled probes,corresponding to each locus,were fixed on CHO-modified glass slide by covalent binding.Hybridization between target sequences and probes was performed under suitable conditions.The spotting locations on microarray and the ratio of fluorescence intensity,produced by different loci,were used to distinguish the SNP genotypes.Finally,three of gastric cancer samples were collected and genotyping analysis for these four SNP loci was carried out successfully simultaneously by this method.     

Multiplexed SNP genotyping using nanobarcode particle technology

Single-nucleotide polymorphisms (SNP) are the most common form of sequence variation in the human genome. Large-scale studies demand high-throughput SNP genotyping platforms. Here we demonstrate the potential of encoded nanowires for use in a particles-based universal array for high-throughput SNP genotyping. The particles are encoded sub-micron metallic nanorods manufactured by electroplating inert metals such as gold and silver into templates and releasing the resulting striped nanoparticles. The power of this technology is that the particles are intrinsically encoded by virtue of the different reflectivity of adjacent metal stripes, enabling the generation of many thousands of unique encoded substrates. Using SNP found within the cytochrome P450 gene family, and a universal short oligonucleotide ligation strategy, we have demonstrated the simultaneous genotyping of 15 SNP; a format requiring discrimination of 30 encoded nanowires (one per allele). To demonstrate applicability to real-world applications, 160 genotypes were determined from multiplex PCR products from 20 genomic DNA samples.     

Electrochemical Genoassay Design for Allele‐Specific Detection of Toll‐Like Receptor‐2 Gene Polymorphism

An allele‐specific voltammetric genoassay for the detection of allele‐specific toll‐like receptor‐2 gene arg753gln polymorphism (TLR‐2) from polymerase chain reaction (PCR) amplified real samples was described in this study. Meldola blue (MDB), an intercalator molecule, was used as hybridization label. The wild‐type and mutant type oligonucleotide probes were immobilized onto disposable graphite electrode surfaces by covalent attachment method. The extent of hybridization between probe and target sequences was determined by using differential pulse voltammetry (DPV). As a result of the interaction between MDB and DNA at electrode surface, the MDB signal observed from probe sequence before hybridization and after hybridization with MM sequence is lower than that observed after hybridization with complementary sequence. The differences between the MDB reduction peaks obtained from probe modified, hybrid modified and MM modified electrode were used to detect TLR‐2 from PCR amplified real samples. The discrimination of homozygous and heterozygous alleles was also established by comparing the peak currents of MDB reduction signals. Numerous factors affecting the target hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

A multiplex SNP genotyping by allele‐specificspecific PCR based on stem‐loop and universal fluorescent primers of Chr1daxin mice

Single nucleotide polymorphisms (SNPs) are one of the most common markers in mammals. Rapid, accurate, and multiplex typing of SNPs is critical for subsequent biological and genetic research. In this study, we have developed a novel method for multiplex genotyping SNPs in mice. The method involves allele‐specific PCR amplification of genomic DNA with two stem‐loop primers accompanied by two different universal fluorescent primers. Blue and green fluorescent signals were conveniently detected on a DNA sequencer. We verified four SNPs of 65 mice based on the novel method, and it is well suited for multiplex genotyping as it requires only one reaction per sample in a single tube with multiplex PCR. The use of universal fluorescent primers greatly reduces the cost of designing different fluorescent probes for each SNP. Therefore, this method can be applied to many biological and genetic studies, such as multiple candidate gene testing, genome‐wide association study, pharmacogenetics, and medical diagnostics.     

High-throughput single nucleotide polymorphism typing by fluorescent single-strand conformation polymorphism analysis with capillary electrophoresis

In this study, we performed high-throughput and precise single nucleotide polymorphism (SNP) typing by fluorescent capillary electrophoresis single-strand conformation polymorphism (CE-SSCP) analysis. A system composed of a multicapillary DNA analyzer, a newly developed sieving matrix, four different colors of fluorescent labels, and a multiplex polymerase chain reaction (PCR) enabled low-cost and highly reliable SNP typing. Moreover, this system enabled the estimation of SNP allele frequencies using pooled DNA samples, which should be beneficial for large-scale association studies. Thus, fluorescent CE-SSCP analysis is a useful method for large-scale SNP typing.     

Rapid genotyping of factor V Leiden mutation using single-tube bidirectional allele-specific amplification and automated ultrathin-layer agarose gel electrophoresis

We report a novel, high-throughput genotyping method by single nucleotide polymorphism (SNP) analysis using bidirectional allele-specific amplification with polymerase chain reaction (PCR) in a single-step/single-tube format. Blood coagulation factor V G1691A (also referred to as Leiden) mutation was chosen as a model system for SNP detection, as this is one of the most common inherited risk factors of thrombosis, effecting 2-5% of the human population. The rationale of our method is the production of allele-specific PCR fragments, different in size, which was achieved by bidirectional amplification, starting from the position of the mutation. Thus, both homozygosity and heterozygosity were readily identified from a single reaction by simply determining the sizes of the resulting PCR products. The advantage of our assay, compared to other single-tube systems, is that this method did not require the use of pre-PCR labeled (fluorophore) primers or probes. Preferential production of the allele-specific products was achieved by a hot-start, time release PCR system. Specificity was increased by introducing a mismatch in the 3'-antepenultimate position of the allele-specific primers. This method made possible the large-scale screening for the factor V Leiden mutation using single-tube PCR followed by automated ultrathin-layer agarose gel electrophoresis, with real-time detection of the "in migratio" ethidium-bromide-labeled fragments.     

Allele specific C-bulge probes with one unique fluorescent molecule discriminate the single nucleotide polymorphism in DNA

A combination of an allele specific C-bulge probe and the fluorescent molecule N,N'-bis(3-aminopropyl)-2,7-diamino-1,8-naphthyridine (DANP) that binds specifically to the C-bulge provides a method for single nucleotide polymorphism (SNP) typing with only one fluorescent molecule without covalent modification of the DNA probe. The allele specific C-bulge probe contains one additional cytosine and produces a C-bulge directly flanking the SNP site upon hybridization to the target DNA. The C-bulge is a scaffold to recruit and retain DANP directly neighboring the SNP site. The DANP fluorescent probe was selectively modulated by the flanking matched and mismatched base pairs. The mutation type could be discriminated by the modulated fluorescent intensity with respect to the allele specific C-bulge probes used for the assay.     

Fungal pathogenic nucleic acid detection achieved with a microfluidic microarray device

Detection of polymerase chain reaction (PCR) products obtained from cultured greenhouse fungal pathogens, Botrytis cinerea and Didymella bryoniae has been achieved using a previously developed microfluidic microarray assembly (MMA) device. The flexible probe construction and rapid DNA detection resulted from the use of centrifugal pumping in the steps of probe introduction and sample delivery, respectively. The line arrays of the oligonucleotide probes were “printed” on a CD-like glass chip using a polydimethylsiloxane (PDMS) polymer plate with radial microfluidic channels, and the sample hybridizations were conducted within the spiral channels on the second plate. The experimental conditions of probe immobilization and sample hybridization were optimized, and both complementary oligonucleotides and PCR products were tested. We were able to achieve adequate fluorescent signals with a sample load as small as 0.5 nM (1 μL) for oligonucleotide samples; for PCR products, we achieved detection at the level of 3 ng.     

A self-assembled deoxyribonucleic acid concatemer for sensitive detection of single nucleotide polymorphism

Polymerase-free and label-free strategies for DNA detection have shown excellent sensitivity and specificity in various biological samples. Herein, we propose a method for single nucleotide polymorphism (SNP) detection by using self-assembled DNA concatemers. Capture probes, bound to magnetic beads, can joint mediator probes by T4 DNA ligase in the presence of target DNA that is complementary to the capture probe and mediator probe. The mediator probes trigger self-assembly of two auxiliary probes on magnetic beads to form DNA concatemers. Separated by a magnetic rack, the double-stranded concatemers on beads can recruit a great amount of SYBR Green I and eventually result in amplified fluorescent signals. In comparison with reported methods for SNP detection, the concatemer-based approach has significant advantages of low background, simplicity, and ultrasensitivity, making it as a convenient platform for clinical applications. As a proof of concept, BRAF^T1799A oncogene mutation, a SNP involved in diverse human cancers, was used as a model target. The developed approach using a fluorescent intercalator can detect as low as 0.1 fM target BRAF^T1799A DNA, which is better than those previously published methods for SNP detection. This method is robust and can be used directly to measure the BRAF^T1799A DNA in complex human serum with excellent recovery (94–103%). It is expected that this assay principle can be directed toward other SNP genes by simply changing the mediator probe and auxiliary probes.