A novel technique for detecting single nucleotide polymorphisms by analyzing consumed allele-specific primers

We present a simple and rapid polymerase chain reaction (PCR)-based technique, termed consumed allele-specific primer analysis (CASPA), as a new strategy for single nucleotide polymorphism (SNP) analysis. The method involves the use of labeled allele-specific primers, differing in length, with several noncomplementary nucleotides added in the 5'-terminal region. After PCR amplification, the amounts of the remaining primers not incorporated into the PCR products are determined. Thus, nucleotide substitutions are identified by measuring the consumption of primers. In this study, the CASPA method was successfully applied to ABO genotyping. In the present method, the allele-specific primer only anneals with the target polymorphic site on the DNA, so it is not necessary to analyze the PCR products. Therefore, this method is only little affected by modification of the PCR products. The CASPA method is expected to be a useful tool for typing of SNPs.     

连接反应介导的等位基因特异性扩增-微流控芯片电泳法同时检测多个SNP位点

以CYP2D6基因中的6个SNP位点为测定对象, 开展多个SNP位点同时测定的方法学研究.     

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.     

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。     

Fluorescence energy transfer-labeled primers for high-performance forensic DNA profiling

A fluorescence energy transfer (ET) dye-labeled STR typing system (ET 16-plex) is developed for the markers used in the commercial STR typing kit PowerPlex 16, and its performance assessed using a 96-lane microfabricated capillary array electrophoresis (muCAE) system. The ET 16-plex amplicons displayed 1.6-9-fold higher fluorescence intensities compared to those produced using the single-dye (SD)-labeled multiplex kits. The ET multiplex delivered full STR profiles from 62.5 pg of DNA; half the input required for the SD kits while maintaining a similar heterozygote allele balance. This increased sensitivity should improve typing of poor-quality DNA samples by making minor or imbalanced alleles more readily detectable at the low copy number (LCN) threshold. The ET 16-plex also generated complete profiles with only 28 PCR cycles; this capability should improve LCN typing by reducing the amplification time and drop-in allele incidence. To confirm the practical advantages of ET-labeled primers, six previously problematic casework samples were tested and only the ET 16-plex kit was able to capture additional allele data. The successful development and demonstration of ET primers for higher sensitivity STR typing offers a simple solution to improving current commercial multiplex typing capability. The superior spectral properties and universal compatibility with any primer sequence provided by ET cassettes will make future multiplex construction more facile and straightforward. The pairing of ET cassette technology with the muCAE system illustrates not only an enhanced STR typing platform, but a significant step toward a higher-efficiency forensic laboratory enabled by better chemistry and microfluidics.     

Single-nucleotide polymorphism analysis by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device

We describe a microfluidic approach for allele-specific extension of fluorescently labeled nucleotides for scoring of single-nucleotide polymorphism (SNP). The method takes advantage of the fact that the reaction kinetics differs between matched and mismatched configurations of allele-specific primers hybridized to DNA template. A microfluidic flow-through device for biochemical reactions on beads was used to take advantage of the reaction kinetics to increase the sequence specificity of the DNA polymerase, discriminating mismatched configurations from matched. The volume of the reaction chamber was 12.5 nL. All three possible variants of an SNP site at codon 72 of the p53 gene were scored using our approach. This work demonstrates the possibility of scoring SNP by allele-specific extension of fluorescently labeled nucleotides in a microfluidic flow-through device. The sensitive detection system and easy microfabrication of the microfluidic device enable further miniaturization and production of an array format of microfluidic devices for high-throughput SNP analysis.     

Rapid single nucleotide polymorphism analysis by primer extension and capillary electrophoresis using polyvinyl pyrrolidone matrix

Rapid molecular diagnosis of 21-hydroxylase deficiency by detecting the most common mutation in the 21-hydroxylase gene is presented using primer extension and capillary electrophoresis with a polyvinyl pyrrolidone matrix. DNA samples were subjected to polymerase chain reaction (PCR) in order to amplify a 422 bp fragment of the CYP21 gene containing the single nucleotide polymorphism (SNP) site. This product served as a template in the primer extension reaction using a fluorescently labeled primer in close proximity to the SNP. ddGTP was used to block the extension if the mutation was present and the other three dNTPs to enable elongation of the primer. Fast analysis of the resulting fragments was performed by capillary electrophoresis using 10% polyvinylpyrrolidone as sieving and wall coating matrix. The Cy5-labeled primer and the two possible primer extension products (mutant and wild type) were completely separated in 90 s.     

基于微流控芯片的72重单核苷酸多态性族群推断系统的构建

建立了常染色体单核苷酸多态性（SNPs）复合检测芯片体系,用于未知个体的族群来源推断。基于前期筛选的74-SNPs组合,采用竞争性等位基因特异性聚合酶链式反应（PCR）的原理构建SNPs的扩增体系,在微流控芯片的每个反应孔内完成一个SNP的检测,通过高通量PCR微流控芯片实现了其中72个SNPs的同步检测。芯片的扩增由平板PCR仪完成,反应孔的荧光信号通过激光共聚焦扫描仪检测,最终通过提取的荧光值进行结果分析。使用该芯片检测获得52份样本的SNPs分型,分型结果的准确率为100%。以57个人群的3628个样本为参考人群数据库,进行20份样本的族群来源推断,推断结果与样本的实际来源一致。本研究建立的常染色体72个SNPs微流控芯片体系可以有效地进行SNP多态性分析检测,基于参考数据库,20份检测样本族群推断的准确性为100%。     

A SNaPshot assay for genotyping 44 individual identification single nucleotide polymorphisms

Single nucleotide polymorphisms (SNPs), which have relatively low mutation rates and can be genotyped after PCR with shorter amplicons compared with short tandem repeats (STRs), are being considered as potentially useful markers in forensic DNA analysis. Those SNPs with high heterozygosity and low Fst (F-statistics) in human populations are described as individual identification SNPs, which perform the same function as STRs used in forensic routine work. In the present study, we developed a multiplex typing method for analyzing 44 selected individual identification SNPs simultaneously by using multiplex PCR reaction in association with fluorescent labeled single base extension (SBE) technique. PCR primers were designed and the lengths of the amplicons ranged from 69 to 125?bp. The population genetics data of 79 unrelated Chinese individuals for the 44 SNP loci were investigated and a series of experiments were performed to validate the characteristic of the SNP multiplex typing assay, such as sensitivity, species specificity and the performance in paternity testing and analysis of highly degraded samples. The results showed that the 44-SNPs multiplex typing assay could be applied in forensic routine work and provide supplementary data when STRs analysis was partial or failed.     

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.     

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.     

Multichannel homogeneous immunoassay for detection of 2,4,6-trinitrotoluene (TNT) using a microfabricated capillary array electrophoresis chip

A high-throughput homogeneous immunoassay for the sensitive detection of 2,4,6-trinitrotoluene (TNT) has been developed using radial capillary array electrophoresis microdevices. Samples consisting of equilibrium mixtures of anti-TNT antibody (Ab), fluorescein-labeled TNT, and various concentrations of unlabeled TNT were electrokinetically injected into 48 channels of a radial capillary array electrophoresis microchannel plate. The rapid electrophoretic separation allows us to analyze the equilibrium ratio formed by the competition between the labeled and the unlabeled TNT for Ab binding. The simultaneous parallel TNT separations facilitate determination of a calibration curve for the TNT assay, which has high sensitivity (LOD, 1 ng/mL) and a wide dynamic range (1-300 ng/mL).     

Integration of gene amplification and capillary gel electrophoresis on a polydimethylsiloxane-glass hybrid microchip

We report on the development of a hybrid polydimethylsiloxane (PDMS)-glass microchip for genetic analysis by functional integration of polymerase chain reaction (PCR) and capillary gel electrophoresis (CGE), and on related temperature control systems for PCR on a PDMS-glass hybrid microchip. The microchip was produced by molding PDMS against a microfabricated master with comparatively simple and inexpensive methods. PCR was successfully carried out on the PDMS-glass hybrid microchip with 500 bp target of lambdaDNA and the amplified gene was subsequently analyzed by CGE on the same PDMS-glass microchip. The chip could be considered as an inexpensive single-use apparatus compared to glass or silicon-made microchips for the same purpose.     

Fully integrated PCR-capillary electrophoresis microsystem for DNA analysis

A fully integrated genomic analysis microsystem including microfabricated heaters, temperature sensors, and PCR chambers directly connected to capillary electrophoretic separation channels has been constructed. Valves and hydrophobic vents provide controlled and sensorless sample positioning and immobilization into 200 nL PCR chambers. The use of microfabricated heating and temperature sensing elements improves the heating and cooling rates for the PCR reaction to 20 degree C s(-1). The amplified PCR product, labeled on-column with an intercalating fluorescent dye, is injected into the gel-filled capillary for electrophoretic analysis. Successful sex determination using a multiplex PCR reaction from human genomic DNA is demonstrated in less than 15 min. This device is an important step toward a microfabricated genomic microprocessor for use in forensics and point-of-care molecular medical diagnostics.     

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.     

Integrated CMOS microchip system with capillary array electrophoresis

A complementary metal oxide semiconductor (CMOS)-capillary array electrophoresis (CAE) system has been used for DNA analysis. Because of its compactness and multiplex capability, the CAE-CMOS microchip is very suitable for the construction of a miniaturized high-throughput system for bioassays. Use of simultaneous laser-beam focusing on to the capillary array and a microscope objective contributed to the construction of the compact CMOS microchip-CAE system. To test the constructed system 100-base-pair (bp) DNA ladders and Hind III digest lambda DNA were separated in poly(vinylpyrrolidone) (PVP) sieving matrix. The miniaturized and integrated CMOS microchip system used in this work had great potential for combination with a variety of microfabricated devices for biomedical research.     

Design of allele-specific primers and detection of the human ABO genotyping to avoid the pseudopositive problem

PCR experiments using DNA primers forming mismatch pairing with template lambda DNA at the 3' end were carried out in order to develop allele-specific primers capable of detecting SNP in genomes without generating pseudopositive amplification products, and thus avoiding the so-called pseudopositive problem. Detectable amounts of PCR products were obtained when primers forming a single or two mismatch pairings at the 3' end were used. In particular, 3' terminal A/C or T/C (primer/template) mismatches tended to allow PCR amplification to proceed, resulting in pseudopositive results in many cases. While less PCR product was observed for primers forming three terminal mismatch pairings, target DNA sequences were efficiently amplified by primers forming two mismatch pairings next to the terminal G/C base pairing. These results indicate that selecting a primer having a 3' terminal nucleotide that recognizes the SNP nucleotide and the next two nucleotides that form mismatch pairings with the template sequence can be used as an allele-specific primer that eliminates the pseudopositive problem. Trials with the human ABO genes demonstrated that this primer design is also useful for detecting a single base pair difference in gene sequences with a signal-to-noise ratio of at least 45.     

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.     

Genotyping of intron 22 inversion of factor VIII gene for diagnosis of hemophilia A by inverse-shifting polymerase chain reaction and capillary electrophoresis

This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40–50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris–borate–EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.     

Preliminary evaluation of electrochemical PNA array for detection of single base mismatch mutations

We report here a preliminary evaluation of a microfabricated disposable-type peptide nucleic acid (PNA) array, with a 20-channel electrode, for the detection of cancer gene c-Ki-ras. Synthetic 15-mer PNA probes complimentary to ras sequence modified with cysteine were immobilized on the gold electrodes on the array. The electrochemical PNA array was reacted with 20-mer oligonucleotide target or 128 bp PCR product for 1 h. The anodic current derived from an electrochemically active DNA binder Hoechst 33258 was measured using the PNA array in the 50 microL of reaction chamber. The anodic current from Hoechst 33258 increased with increasing the concentration of PCR product of ras gene in the range from 10(11) to 10(15) copy mL(-1). The single base mismatch mutations of c-Ki-ras/61 were also detected using the electrochemical PNA array.