Use of an electrochemically labeled nucleotide terminator for known point mutation analysis

A novel single nucleotide polymorphism (SNP) assay utilizing an electrochemically tagged chain terminator is described. The system employs the single-base extension (SBE) technique coupled to capillary gel electrophoresis with end-column electrochemical detection. A redox-labeled chain terminator, ferrocene-acycloATP, is used in the SBE reaction. When the mutation site corresponds to the labeled chain terminator, the extension product is rendered electroactive. The reaction mixture is subsequently separated by capillary gel electrophoresis and the extension product detected at the separation anode with sinusoidal voltammetry. This work demonstrates the first known SNP assay utilizing redox-active chain terminators coupled to electrochemical detection. The methodology presented could lead to a fast, simple, and cost-effective SNP scoring system.     

Genotyping of single nucleotide polymorphism in MDM2 genes by universal fluorescence primer PCR and capillary electrophoresis

Single nucleotide polymorphism (SNP) 309 in the promoter region of the murine double minute 2 (MDM2) gene plays an important role in human tumorigenesis. We established a simple and effective CE method for SNP detection in the MDM2 gene. We designed one universal fluorescence-based nonhuman-sequence primer and one fragment-oriented primer, which were combined in one tube, and proceeded with the polymerase chain reaction (PCR). The amplicons were analyzed by capillary electrophoresis using single-strand conformation polymorphism method. PCR fragments generated from this two-in-one PCR displayed either T/T or G/G homozygosity or T/G heterozygosity. A total of 304 samples were blindly genotyped using this developed method, which included the DNA from 138 healthy volunteers, 43 chronic myeloid leukemia (CML) patients, and 123 colorectal cancer (CRC) patients. The results were confirmed by DNA sequencing and showed good agreement. The SSCP-CE method was feasible for SNP screening of MDM2 in large populations.     

Multiplex high-throughput solid-phase minisequencing by capillary electrophoresis and liquid core waveguide fluorescence detection

Minisequencing, solid-phase single-nucleotide primer extension reaction, is a robust method for performing multiplex single-nucleotide polymorphism (SNP) analysis. We have combined this technology with capillary gel electrophoresis in a multicapillary format, using liquid core waveguide (LCW) fluorescence detection. Polymerase chain reaction (PCR) amplification of multiple DNA targets is performed with one primer for each target biotinylated. Separation of the complementary strands, minisequencing and washing steps are carried out using streptavidin-coated magnetic beads. Dideoxynucleotides analogues labelled with different fluorophores are used for the extension of the minisequencing primers. The extended oligonucleotides, the length of which defines the position on the target and the color the identity of the polymorphism, are then separated in a gel-filled array of capillaries, coated on the outside with a layer of a fluoropolymer to provide the liquid core waveguide characteristics. The technology has a potential for extremely high throughputs when a combination of multiplex PCR-minisequencing is used together with a large array of capillaries, four-color detection and high-speed separation.     

High speed single nucleotide polymorphism typing of a hereditary haemochromatosis mutation with capillary array electrophoresis microplates

A single nucleotide polymorphism (SNP) typing assay is developed and evaluated on a microfabricated capillary array electrophoresis system. Using fluorescently labeled allele-specific primers, the S65C (193A-->T) substitution associated with hereditary haemochromatosis in the HFE gene is genotyped. The covalently labeled polymerase chain reaction (PCR) products are separated on a microfabricated radial capillary array electrophoresis microplate using nondenaturing gel media in under two minutes. Detection is accomplished with a laser-excited rotary confocal scanner. The Rox-labeled A-allele specific amplicon (211 bp) is differentiated from the R110-labeled T-allele specific amplicon (201 bp) by both size and color. This study demonstrates the feasibility of using allele-specific PCR with covalently labeled primers for high speed fluorescent SNP typing on microfabricated radial capillary array electrophoresis microplates.     

毛细管电泳——基因突变及多态性分析新方法

着重介绍基因突变及多态性分析方法以及毛细管在电泳在该领域中的应用。主要包括单链构象多态性分析,变性梯度及温度梯度电泳,杂合子分析,限制性片段多态性分析,等位基因特异性扩增,核酸杂交,引物扩展及小卫星和微卫星分析。     

Effect of polymer matrix and glycerol on rapid single-strand conformation polymorphism analysis by capillary and microchip electrophoresis for detection of mutations in K-ras gene

We present the rapid single-strand conformation polymorphism (SSCP) analysis by capillary and microchip electrophoresis to detect the mutations in K-ras gene. Parameters that might affect the analysis of mutation in K-ras gene, such as the polymer and the additive in the sieving matrix, have been studied systematically. Under the optimal conditions, the analysis of seven mutants of K-ras gene could be finished within 10 min by capillary electrophoresis (CE). Furthermore, with the wild-type gene as the inner standard, the analysis accuracy of mutations could be improved. In addition, by studying the properties of polymer solutions, the matrix suitable for microchip electrophoresis was found, and the detection of mutations in K-ras gene could be further shortened to 1 min.     

Single nucleotide polymorphism analysis by allele-specific primer extension with real-time bioluminescence detection in a microfluidic device

A microfluidic approach for rapid bioluminescent real-time detection of single nucleotide polymorphism (SNP) is presented. The method is based on single-step primer extension using pyrosequencing chemistry to monitor nucleotide incorporations in real-time. The method takes advantage of the fact that the reaction kinetics differ between matched and mismatched primer-template configurations. We show here that monitoring the initial reaction in real time accurately scores SNPs by comparing the initial reaction kinetics between matched and mismatched configurations. Thus, no additional treatment is required to improve the sequence specificity of the extension, which has been the case for many allele-specific extension assays. The microfluidic approach was evaluated using four SNPs. Three of the SNPs included primer-template configurations that have been previously reported to be difficult to resolve by allele-specific primer extension. All SNPs investigated were successfully scored. Using the microfluidic device, the volume for the bioluminescent assay was reduced dramatically, thus offering a cost-effective and fast SNP analysis method.     

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.     

BRCA1 mutation screening using restriction endonuclease fingerprinting-single-strand conformation polymorphism in an automated capillary electrophoresis system

Efficient mutation scanning techniques are needed for the rapid detection of novel disease-associated mutations and rare-sequence variants of putative importance. The large size of the breast cancer 1 gene (BRCA1) and the many mutations found throughout its entire coding sequence make screening for mutations in this gene particularly challenging. We have developed a method for screening exon 11 of the BRCA1 gene based on restriction enzyme digestion of fluorescence-labeled polymerase chain reaction (PCR) products followed by single-strand conformation polymorphism (SSCP) using an automated capillary electrophoresis system, denoted capillary restriction endonuclease fingerprinting (REF)-SSCP electrophoresis. Using this strategy on a control set of samples, we were able to detect 17 of 18 known sequence alterations. The method was then applied to screen 73 Norwegian females with family histories of breast and/or ovarian cancer. A total of 172 sequence alterations were detected, including substitutions, insertions, and deletions. One novel substitution of unknown function was identified. Sequencing of all samples negative in the capillary REF-SSCP system gave no additional mutations confirming the high sensitivity of the described methodology. Capillary REF-SSCP electrophoresis appeared as a technically convenient technique, requiring amplification of fewer PCR fragments than traditional SSCP. The novel strategy allows high-throughput mutation scanning without radioactive labeling and polyacrylamide gel electrophoresis (PAGE).     

Microchip capillary gel electrophoresis with electrochemical detection for the analysis of known SNPs

A novel microchip-based single nucleotide polymorphism (SNP) screening system has been developed. The system utilizes capillary gel electrophoresis (CGE) with electrochemical detection in a chip-based format to accomplish rapid scoring of a mock SNP site. The accuracy of the thermostable polymerase and the advantages of coupling this technique to microfluidics are demonstrated. An electrochemically labeled chain terminator is used in the single base extension (SBE) reaction, in which the terminator is incorporated only when its Watson-Crick complementary base is present at the mock SNP site. The resulting electrochemically active extension product is subsequently separated from any excess terminator by CGE and detected by sinusoidal voltammetry. Although no attempts at optimization have been made, the analysis is performed in less than 4 min. The technique presented could lead to a fast, simple, and cost effective SNP scoring system.     

毛细管电泳-限制性片段长度多态性分析检测胃癌H-ras基因点突变

建立一种毛细管电泳快速高效检测限制性内切酶酶切产物的方法, 使其更好地用于基因诊断. 以甲基纤维素(Methyl cellulose, MC)为筛分介质, 用pUC19 DNA/Msp I (Hpa II) Marker标准DNA片段为实验对象, 通过考察筛分介质的浓度、pH值、毛细管的温度和运行电压优化出分离小于600 bp的双链DNA片段的最适条件, 并将此方法应用于临床59例胃癌患者肿瘤组织H-ras基因12位密码子点突变情况的检测. MC是一种良好的筛分介质, 运用其进行毛细管电泳对于遗传性疾病的诊断将更加快速、准确、简便、灵敏.     

Analysis of single nucleotide polymorphisms by primer extension and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method for typing single nucleotide polymorphisms (SNPs) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) is described, in which a mass-tagged dideoxynucleoside triphosphate is employed in a primer extension reaction in place of an unmodified dideoxynucleoside triphosphate (ddNTP). The increased mass difference due to the presence of the mass-tag greatly facilitates the accurate identification of the added nucleotide, and is particularly useful for typing heterozygous samples. Twenty commercially available mass-tagged dideoxynucleoside triphosphates were screened for amenability to incorporation by AmpliTaq FS and ThermoSequenase DNA polymerases in single nucleotide primer extension (SNuPE) reactions. Several sample preparation and purification methods were also examined and compared. Float dialysis was found to be a simple, versatile, and effective method for purification of the extension products. High specificity and sensitivity were obtained, and all six possible biallelic SNP heterozygotes were determined accurately using a 44-mer synthetic oligonucleotide target DNA as a model system. Further validation of the method was demonstrated in the analysis of five single-base mutations in exon IV of the human tyrosinase gene. Single nucleotide variations within 182-bp PCR amplicons amplified from three plasmid and three human genomic DNA samples were genotyped at five variable positions, with results in 100% concordance with conventional sequencing. Genotypes were determined accurately at five sequence-tagged sites (STSs).     

Label-free genotyping of single-nucleotide polymorphisms for DNA and RNA targets using a cationic polythiophene derivate

By using the specific primer extension reaction, a new assay for genotyping of single-nucleotide polymorphisms (SNPs) has been demonstrated. The assay relies on the conformational and colorimetric change of water-soluble polythiophene derivative, poly[3-(3′-N,N,N-triethylamino-1′-propyloxy)-4-methyl-2,5-thiophene hydrochloride] (PMNT), upon forming interpolyelectrolyte complex with extended double strand DNA and non-extended single strand DNA. All three kinds of SNP genotypes can be colorimetrically identified with one primer extension reaction in homogeneous solution. Moreover, combining with the specific digestion of RNA strands in the RNA/DNA hybrids, the proposed assay can also be applied to SNP genotyping for RNA templates. The SNP genotyping assay does not require chemical modification of oligonucleotide probes and nucleic acid targets and any separation step. It would be useful for routinely SNP detection in ordinary laboratories.     

微流控芯片电泳-多重聚合酶链反应法同时测定多个单碱基多态性位点

以微流控芯片电泳为检测平台,建立了多重PCR扩增法同时测定多个单碱基多态性(SNP)位点的方法。先通过PCR扩增得一段含所有待测SNP位点的长片段;用限制性内切酶消化成短片段,再将酶切反应产物与脱氧核糖核酸适配器(DNAadapter)相连;以连接产物为模板,分成两管,分别用n条等位基因特异性引物和一条通用引物进行n重PCR扩增;最后用微流控芯片电泳法分离PCR扩增产物,根据两管扩增产物的芯片电泳图谱中扩增片段的大小判断SNP的类型。以细胞色素P4502D6(CYP2D6)基因中的5个SNP位点(100C>T、1661G>C、1758G>T、2470T>C和2850C>T)为检测对象,考察了各等位基因特异性引物之间的相互影响和扩增反应的特异性,采用微流控芯片电泳法成功测定了20名健康中国人的CYP2D6基因中5个SNP位点的基因多态性,与聚合酶链反应-限制性片段长度多态性法(PCR-RFLP)测定结果完全一致。     

Genetic mutation analysis by CE with LIF detection using inverse-flow derivatization of DNA fragments

Inverse-flow derivatization is a novel approach to obtain fluorescent DNA derivatives in DNA analysis based on CE with LIF detection. In the present work, we want to explore the feasibility of the application of this method into the mutation detection based on constant denaturant capillary electrophoresis (CDCE) and SSCP analysis. The DNA fragments were first amplified by PCR using a pair of common primers without fluorescent label, and then the mutations were determined by CDCE or SSCP analysis based on CE-LIF with inverse-flow derivatization of DNA fragments. The experimental conditions were investigated systematically, and different labeling modes including inverse-flow derivatization, on-column derivatization and fluorescent labeled primer technique were compared. The inverse-flow derivatization was successfully used in the detection of C677T mutation in the methylenetetrahydrofolate reductase gene by CDCE or SSCP analysis. Our preliminary results demonstrate that inverse-flow derivatization is very simple, inexpensive and sensitive and well suitable for the genetic analysis in clinical diagnosis.     

Fluorescence-based single-strand conformation polymorphism analysis of mutations by capillary electrophoresis

Capillary electrophoresis in combination with fluorescence-based single-strand conformation polymorphism (SSCP) analysis was used to screen for known mutations as well as for unknown mutations. The mutations causing hemochromatosis and thrombogenetic diseases (factor V Leiden mutation and prothrombin mutation) are well defined. Familial hypercholesterolemia is caused by mutations in the low density lipoprotein (LDL) receptor gene. Because the mutations are heterogeneously localized in all 18 exons of the LDL receptor gene, effective screening procedures are necessary. The three well known mutations and 59 of 61 previously characterized mutations in the LDL receptor gene were detected by a distinct abnormal fragment pattern in capillary electrophoresis. The remaining two mutations in the LDL receptor gene showed only slight abnormalities under standard electrophoresis conditions (13 kV, 30 degrees C, 30 min). However, the abnormal pattern could be amplified by increasing the electrophoresis temperature. In all cases, heterozygous and homozygous mutations could clearly be differentiated from wild-type alleles. Because of the high efficiency of mutation detection, capillary electrophoresis in combination with fluorescence-based SSCP analysis would be attractive for the detection of well-defined mutations as well as for the screening of unknown mutations. The accuracy and the degree of automation make this technique well suited for routine genetic diagnosis.     

DNA Dangling‐End‐Induced Colloidal Stabilization of Gold Nanoparticles for Colorimetric Single‐Nucleotide Polymorphism Genotyping

A single‐nucleotide polymorphism (SNP) detection method was developed by combining single‐base primer extension and salt‐induced aggregation of gold nanoparticles densely functionalized with double‐stranded DNA (dsDNA‐AuNP). The dsDNA‐AuNPs undergo rapid aggregation in a medium of high ionic strength, whereas particles having a single‐base protrusion at the outermost surface disperse stably, allowing detection of a single‐base difference in length by color changes. When SNP typing primers are used as analytes to hybridize to the single‐stranded DNA on the AuNP surface, the resulting dsDNA‐AuNP works as a visual indicator of single‐base extension. A set of four extension reaction mixtures is prepared using each of ddNTPs and subsequently subjected to the aggregation assay. Three mixtures involving ddNTP that is not complementary to the SNP site in the target produce the aggregates that exhibit a purple color. In contrast, one mixture with the complementary ddNTP generates the single‐base protrusion and appears red. This method could potentially be used in clinical diagnostics for personalized medicine.     

Detection of point mutations by capillary electrophoresis with temporal temperature gradients.

By combining the advantages of capillary electrophoresis and temperature gradient gel electrophoresis, a method was developed to detect point mutations in polymerase chain reaction (PCR) fragments. Increasing and decreasing temporal temperature gradients were established by means of a computer-controlled Peltier module. Native and denaturing conditions were achieved by cooling to 25 degrees C and heating to 70 degrees C, respectively, a thermostating liquid surrounding the capillary. To separate nucleic acid fragments, a sieving media, containing 4% linear polyacrylamide, 1 x Tris borate EDTA buffer (TBE) and 6 M urea, was found appropriate. Renewal of the sieving matrix before each run significantly improved the reproducibility of fragment separation. The ability of this capillary electrophoresis system to detect point mutations is demonstrated with the human prion-protein gene.     

Capillary electrophoresis as a tool for optimization of multiplex PCR reactions

Copying multiple regions of a DNA molecule is routinely performed today using the polymerase chain reaction (PCR) in a process commonly referred to as multiplex PCR. The development of a multiplex PCR reaction involves designing primer sets and examining various combinations of those primer sets and different reaction components and/or thermal cycling conditions. The process of optimizing a multiplex PCR reaction in order to obtain a well-balanced set of amplicons can be time-consuming and labor-intensive. The rapid separation and quantitation capabilities of capillary electrophoresis make it an efficient technique to help in the multiplex PCR optimization process.