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 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.     

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.     

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.     

Rapid discrimination of single-nucleotide mismatches using a microfluidic device with monolayered beads

A microfluidic device incorporating monolayered beads is developed for the discrimination of single-nucleotide mismatches, based on the differential dissociation kinetics between perfect match (PM) and mismatched (MM) duplexes. The monolayered beads are used as solid support for the immobilization of oligonucleotide probes containing a single-base variation. Target oligonucleotides hybridize to the probes, forming either PM duplexes or MM duplexes containing a single mismatch. Optimization studies show that PM and MM duplexes are easily discriminated based on their dissociation but not hybridization kinetics under an optimized buffer composition of 100 mM NaCl and 50% formamide. Detection of single-nucleotide polymorphism (SNP) using the device is demonstrated within 8 min using four probes containing all the possible single-base variants. The device can easily be modified to integrate multiplexed detection, making high-throughput SNP detection possible.     

A novel crossed microfluidic device for the precise positioning of proteins and vesicles

Herein we present a novel way to create arrays of different proteins or lipid vesicles using a crossed microfluidic device. The concept relies on the combination of I) a designated two-step surface chemistry, which allows activation for subsequent binding events, and II) crossing microfluidic channels for the local functionalization by separated laminar streams. Besides its simplicity and cost efficiency, this concept has the advantage that it keeps the proteins in a hydrated environment throughout the experiment. We have demonstrated the feasibility of such a device to create a chessboard pattern of different fluorescently labeled lipid vesicles, which offers the possibility to contain biomolecules, drugs or membrane proteins.     

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%。     

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.     

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.     

Immobilization and detection of DNA on microfluidic chips

With the rapid development of micro-Total Analysis Systems (muTAS) and sensitive DNA recognition technologies, it is possible to immobilize DNA probes to small areas of surfaces other than silicon. To this end, photolithographic techniques were used to derivatize micron-sized, spatially segregated DNA recognition elements in Polydimethylsiloxane (PDMS) microfluidic structures. UV light was used to initiate attachment of a photoactive biotin molecule to the substrate surface. Once biotin was attached to a substrate, biotin/avidin/biotin chemistry was used to attach fluorescently labeled or non-labeled avidin and biotinylated DNA probes. These techniques were applied to create a prototype microfluidic sensor device that was used to separate and identify synthetic DNA targets that were fluorescently-labeled.     

Increased single-nucleotide discrimination in allele-specific polymerase chain reactions through primer probes bearing nucleobase and 2'-deoxyribose modifications

The diagnosis of genetic dissimilarities between individuals is becoming increasingly important due to the discovery that these variations are related to complex phenotypes like the predisposition to certain diseases or compatibility with drugs. The most common among these sequence variations are single-nucleotide polymorphisms (SNPs). The availability of reliable and efficient methods for the interrogation of the respective genotypes is the basis for any progress along these lines. Many methods for the detection of nucleotide variations in genes exist, in which amplification of the target gene is required before analysis can take place. The allele-specific polymerase chain reaction (asPCR) combines target amplification and analysis in a single step. The principle of asPCR is based on the formation of matched or mismatched primer-target complexes. The most important parameter in asPCR is the discrimination of these matched or mismatched pairs. In recent publications we have shown that the reliability of SNP detection through asPCR is increased by employing chemically modified primer probes. In particular, primer probes that bear a polar 4'-C-methoxymethylene residue at the 3' end have superior properties in discriminating single-nucleotide variations by PCR. Here we describe the synthesis of several primer probes that bear nucleobase modifications in addition to the 4'-C-methoxymethylenated 2'-deoxyriboses. We studied the effects of these alterations on single-nucleotide discrimination in allele-specific PCR promoted by a DNA polymerase and completed these results with single-nucleotide-incorporation kinetic studies. Moreover, we investigated thermal denaturing of the primer-probe-template complexes and recorded circular dichroism (CD) spectra for inspecting the thermodynamic and photophysical duplex behaviour of the introduced modifications. In short, we found that primer probes bearing a 4'-C-methoxymethylene residue at the 2'-deoxyribose moiety in combination with a thiolated thymidine moiety have synergistic effects and display significantly increased discrimination properties in asPCR.     

Serial processing of biological reactions using flow-through microfluidic devices: coupled PCR/LDR for the detection of low-abundant DNA point mutations

We have fabricated a flow-through biochip consisting of passive elements for the analysis of single base mutations in genomic DNA using polycarbonate (PC) as the substrate. The biochip was configured to carry out two processing steps on the input sample, a primary polymerase chain reaction (PCR) followed by an allele-specific ligation detection reaction (LDR) for scoring the presence of low abundant point mutations in genomic DNA. The operation of the device was demonstrated by detecting single nucleotide polymorphisms in gene fragments (K-ras) that carry high diagnostic value for colorectal cancers. The effect of carryover from the primary PCR on the subsequent LDR was investigated in terms of LDR yield and fidelity. We found that a post-PCR treatment step prior to the LDR phase of the assay was not essential. As a consequence, a thermal cycling microchip was used for a sequential PCR/LDR in a simple continuous-flow format, in which the following three steps were carried out: (1) exponential amplification of the gene fragments from genomic DNA; (2) mixing of the resultant PCR product(s) with an LDR cocktail via a Y-shaped passive micromixer; and (3) ligation of two primers (discriminating primer that carried the complement base to the mutation locus being interrogated and a common primer) only when the particular mutation was present in the genomic DNA. We successfully demonstrated the ability to detect one mutant DNA in 1000 normal sequences with the integrated microfluidic system. The PCR/LDR assay using the microchip performed the entire assay at a relatively fast processing speed: 18.7 min for 30 rounds of PCR, 4.1 min for 13 rounds of LDR (total processing time = ca. 22.8 min) and could screen multiple mutations simultaneously in a multiplexed format. In addition, the low cost of the biochip due to the fact that it was fabricated from polymers using replication technologies and consisted of passive elements makes the platform amenable to clinical diagnostics, where one-time use devices are required to eliminate false positives resulting from carryover contamination.     

Detection of single-nucleotide polymorphisms coding for three ovine prion protein variants by primer extension assay and capillary electrophoresis

An alternative method is described for the determination of ovine prion protein allelic variants at codon 136, 154, and 171. The four mutations responsible for amino acid changes are typed simultaneously. The technique utilizes dideoxy chain termination reaction using fluorescently labeled dideoxy nucleotides. The single-base extended primers are resolved on a capillary electrophoresis instrument. Data obtained by our approach are presented according to genotype distribution in some breeds as a part of the validation procedure.     

An integrated method for mutation detection using on-chip sample preparation, single-stranded conformation polymorphism, and heteroduplex analysis

This work integrates rapid techniques for mutation detection by producing single-stranded DNA and (renatured) double-stranded DNA on-chip, labeling these with fluorescent DNA stains and then performing two complementary methods of mutation detection-single stranded conformation polymorphism (SSCP) analysis and heteroduplex analysis (HA). This involves the denaturation of double-stranded polymerase chain reaction (PCR) product into single-stranded DNA, the mutation analysis of the single-stranded DNA by SSCP and the rehybridized double-stranded DNA by HA. These steps were performed entirely on-chip within several minutes of operation. The combination of these two mutation detection methods on-chip provides a highly sensitive method of mutation detection for either genotyping or screening. Many mutation analysis methods rely upon fluorescently labeled samples from a PCR with fluorescently labeled primers. By labeling on-chip we not only attain improved signal strength, but the method is considerably more versatile. Although we used PCR products in this work, this method could be used to analyze DNA from any source. We believe that this combination of several procedures on a single chip represents a significant step in the development of higher levels of integration upon microfluidic devices.     

水溶性碳纳米粒子的制备及其在DNA和单核苷酸多态性分析中的应用

利用电化学氧化的方法制备了水溶性好、粒径为7～12nm的碳纳米粒子,该碳纳米粒子通过π-π相互作用吸附荧光标记的单链DNA探针,并能有效地猝灭其荧光．当单链DNA探针与匹配的DNA目标分子杂交形成双链DNA时,猝灭的荧光被恢复,由此可以检测1-200nmol／L的DNA目标分子。此外,在碳纳米粒子存在时,由荧光标记的DNA探针和DNA目标分子形成的双链DNA的熔解温度可以简便地被测定,当双链DNA有错配碱基时,其熔解温度降低,由此可方便、快速地分析单核苷酸多态性．     

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.     

Human Y-chromosome haplotyping by allele-specific polymerase chain reaction

We describe the application of allele-specific PCR (AS-PCR) for screening biallelic markers, including SNPs, within the nonrecombining region of the human Y-chromosome (NRY). The AS-PCR method is based on the concept that the perfectly annealed primer-template complex is more stable, and therefore, more efficiently amplified under the appropriate annealing temperature than the complex with a mismatched 3'-residue. Furthermore, a mismatched nucleotide at the primer's 3'-OH end provides for a poor extension substrate for Taq DNA polymerase, allowing for discrimination between the two alleles. This method has the dual advantage of amplification and detection of alleles in a single expeditious and inexpensive procedure. The amplification conditions of over 50 binary markers, mostly SNPs, that define the major Y-haplogroups as well as their derived lineages were optimized and are provided for the first time. In addition, artificial restriction sites were designed for those markers that are not selectively amplified by AS-PCR. Our results are consistent with allele designations derived from other techniques such as RFLP and direct sequencing of PCR products.     

Integrated self-calibration via electrokinetic solvent proportioning for microfluidic immunoassays.

On-board generation of a set of calibration standards was demonstrated within a microfluidic device designed to perform immunoassay. Electrokinetic flow was used to proportionally mix the antibody (Ab) to bovine serum albumin (BSA) and a diluting buffer, to provide varying Ab concentrations for downstream mixing with fluorescently labeled BSA (BSA*). Mixing ratios were determined from electrical impedance modeling of the fluidic network using P-SPICE software, and peak heights for the labeled species were analyzed relative to the concentration calculated from the model. For dilution and separation of fluorescently labeled amino acids, a linear calibration curve was obtained for mixing ratios of 0.118 to 7.46. A linear calibration curve was obtained for the immunoassay calibration using dilution ratios between 0.197 and 5.077. Deviations were observed at larger extremes, possibly due to leakage effects at intersections. Peak height reproducibility was +/- 3% for the immunoassay, using diluted monoclonal Ab in mouse ascites fluid as the analyte. Recovery for on-chip calibration was 92 +/- 6% versus calibrants prepared off-chip, indicating a small bias.     

Hybridization of mismatched or partially matched DNA at surfaces

We investigate how probe density influences hybridization for unlabeled target oligonucleotides that contain mismatched sequences or targets that access different binding locations on the immobilized probe. We find strong probe density effects influencing not only the efficiency of hybridization but also the kinetics of capture. Probe surfaces are used repeatedly, and the potentially large contributions of sample-to-sample variations in surface heterogeneity and nonspecific adsorption are addressed. Results of kinetic, equilibrium, and temperature-dependent studies, obtained using in-situ surface plasmon resonance (SPR) spectroscopy, show that hybridization for surface immobilized DNA is quite different from the well-studied solution-phase reaction. Surface hybridization depends strongly on the target sequence and probe density. Much of the data can be explained by the presence of steric crowding at high probe density; however, the behavior of mismatched sequences cannot be understood using standard models of hybridization even at the lowest density studied. In addition to unusual capture kinetics observed for the mismatched targets, we find that the binding isotherms can be fit only if a heterogeneous model is used. For mismatched targets, the Sips model adequately describes probe-target binding isotherms; for perfectly matched targets, the Langmuir model can be used.     

Interfacial stabilization of organic-aqueous two-phase microflows for a miniaturized DNA extraction module

Organic-aqueous liquid (phenol) extraction is one of many standard techniques to efficiently purify DNA directly from cells. The cell components naturally distribute themselves into the two fluid phases in order to minimize interaction energies of the biological components with the surrounding solvents. The membrane components and protein partition to the interface between the organic and aqueous phases while the DNA stays in the aqueous phase. The aqueous phase is then removed with a purified DNA sample. This work studies the first steps towards miniaturizing this liquid extraction technique in a microfluidic device. The first step is to understand how the two liquid phases behave in microchannels. Due to the interfacial tension between the two liquid phases, novel approaches must be examined in order to obtain interfacial stability under flow conditions. The stability of the organic-aqueous interface is improved by reducing the interfacial tension between the two phases by incorporating a surfactant into the aqueous phase. The variation of the interfacial tension as a function of surfactant concentration is also quantified in this work. This has led to the ability to create stable stratified microflows in both a dual inlet and three inlet microfluidic systems. Also, the first step in understanding biological interactions at the organic-aqueous interface is investigated using a fluorescently labeled bovine serum albumin protein.