DNA microsatellite analysis using ion-pair reversed-phase high-performance liquid chromatography

Genotyping based on short tandem repeat (STR) regions is used in human identification and parentage testing, gene mapping studies, cancer diagnostics, and diagnosis of hereditary diseases. Analysis of STR systems using slab gel electrophoresis requires lengthy and labor-intensive procedures. Therefore, alternative methods such as capillary electrophoresis or ion-pair reversed-phase high-performance liquid chromatography (IPRP HPLC) have been used to analyze DNA. IPRP HPLC offers an attractive substitute to gel electrophoresis for STR analysis because of the reduced analysis time, and there is no need for the waste disposal associated with radioisotopic, enzyme-linked, or fluorescence detection systems. We evaluated the use of IPRP HPLC for the sizing and typing of STR alleles from the HUMTHO1 locus. The IPRP HPLC conditions (column temperature, flow rate, percent organic modifier per minute) were optimized for the separation of PCR products. Using the optimized separation conditions, the alleles of the HUMTHO1 system were sized in their native state (double standard) with the use of internal markers. The typing results correlated 100% to accepted methods of DNA typing. The analysis time for the HUMTHO1 locus was less than 14 min, and the alleles could be peak captured for further examination following such as sequencing.     

Integrated portable genetic analysis microsystem for pathogen/infectious disease detection

An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.     

NIST Mixed Stain Study 3: DNA quantitation accuracy and its influence on short tandem repeat multiplex signal intensity

The Mixed Stain Study 3 (MSS3) interlaboratory challenge exercise evaluated the 2001 performance of STR multiplex DNA typing systems using a set of seven DNA extracts of designed concentration and composition. This initial report focuses on the linkages connecting the measurement of the concentration of DNA ([DNA]) to the observed STR multiplex signal intensities. There is a causal relationship between [DNA] measurement accuracy and the efficiency of STR multiplex analysis. There are no intrinsic measurement performance differences among the [DNA] measurement technologies reported. However, there are large differences in the efficiencies of amplification, separation, and detection among participants using the same nominal measurement systems.     

Analysis of genetic markers in forensic DNA samples using the polymerase chain reaction.

The ability to extract and type DNA from forensic evidentiary samples has revolutionized the field of forensic serology. Previously, genetic marker typing was limited to the analysis of blood group markers and soluble polymorphic protein markers. Because the number of suitable markers expressed in particular fluids and tissues is relatively small, and because mixtures of fluids cannot be separated for conventional genetic marker typing, a suspect frequently cannot be included or excluded as a fluid donor in a case. However, the development of methods to extract DNA from virtually all biological specimens has greatly expanded the potential for individual identification. Of particular importance was the ability to extract mixtures of sperm cells and epithelial cells found in sexual assault cases such that the DNA from the sperm cells could be typed independently of the DNA from the victim's epithelial cells. Restriction fragment length polymorphism (RFLP) analysis was the first DNA-based method applied to problems of individual identification. This method, while powerful in its ability to differentiate individuals, is limited by the quantity and quality of DNA required for an unambiguous result and by the amount of time it takes to obtain a result. Despite these limitations, several laboratories are using RFLP analysis successfully for the detection of polymorphisms in forensic DNA case samples. While the field of forensic serology was being revolutionized by the prospect of DNA analysis, the field of molecular biology was being revolutionized by the invention of the polymerase chain reaction (PCR), which ultimately has had an impact on every area of biological science. The PCR DNA amplification technology is ideally suited for the analysis of forensic DNA samples in that it is sensitive and rapid and not as limited by the quality of DNA as the RFLP method. The focus of this article is the use of the PCR for typing genetic markers, and we will address specifically the special considerations that arise from applying DNA amplification and typing technology to forensic materials.     

A battery-powered notebook thermal cycler for rapid multiplex real-time PCR analysis

A compact, real-time PCR instrument was developed for rapid, multiplex analysis of nucleic acids in an inexpensive, portable format. The instrument consists of a notebook computer, two reaction modules with integrated optics for four-color fluorescence detection, batteries, and a battery-charging system. The instrument weighs 3.3 kg, measures 26 x 22 x 7.5 cm, and can run continuously on the internal batteries for 4 h. Independent control of the modules allows differing temperature profiles and detection schemes to be run simultaneously. Results are presented that demonstrate rapid (1) detection and identification of Bacillus subtilis and Bacillus thuringensis spores and (2) characterization of a single nucleotide polymorphism for the hereditary hemochromatosis gene.     

Laser-induced fluorescence detection by liquid core waveguiding applied to DNA sequencing by capillary electrophoresis

A new laser-induced fluorescence detector for capillary electrophoresis (CE) is described. The detector is based on transverse illumination and collection of the emitted fluorescent light via total internal reflection along the separation capillary. The capillary is coated with a low refractive index fluoropolymer and serves as a liquid core waveguide (LCW). The emitted light is detected end-on with a CCD camera at the capillary exit. The observed detection limit for fluorescein is 2.7 pM (550 ymol) in the continuous-flow mode and 62 fM in the CE mode. The detector is applied to DNA sequencing. One-color G sequencing is performed with single-base resolution and signal-to-noise ratio approximately 250 for peaks around 500 bases. The signal-to-noise ratio is approximately 50 for peaks around 950 bases. Full four-color DNA sequencing is also demonstrated. The high sensitivity of the detector is suggested to partly be due to the efficient rejection of scattered laser light in the LCW. The concept should be highly suitable for capillary array detection.     

法医DNA标准物质STR位点等位基因分型定值研究

为探讨法医DNA标准物质STR位点等位基因分型的定值和溯源,利用有机法提取法医DNA标准物质备选所用细胞基因组DNA,以其为模板进行STR复合扩增,制备获得等位基因分型片段并测序分析,成功获得法医DNA标准物质备选所用细胞STR位点等位基因分型的定值。对STR位点等位基因分型进行定值研究可以作为法医DNA标准物质溯源研究的有效途径之一。     

Dual-view imaging system using a wide-range dichroic mirror for simultaneous four-color single-molecule detection

A dual-view imaging system for simultaneous four-color single-molecule (SM) detection was developed. As for the detection procedure, four species of SM fluorophores, namely, Alexa 488, 555, 647, and 680, are immobilized on different slides and excited by evanescent-wave illumination. Fluorescence emitted from an SM fluorophore is split by a wide-range dichroic mirror (WR DM) in a dual-view optics and imaged as two SM fluorescence spots (SM spots) on an electron-multiplying charge-coupled device (EM-CCD) at 100 Hz. The transmittance of the WR DM changes gradually over the wavelength range of 500 to 700 nm so that the signal ratios of the two SM spots for the four fluorophore species differ. A method for classifying SM fluorophores into four species in accordance with their signal ratios was developed. It was used to classify 597 SM fluorophores at an accuracy of above 98% for all the species. This accuracy is comparable to that of a conventional four-color SM detection system. To classify four species, the conventional system disperses SM fluorescence with a prism and provides an elongated SM spot that uses more pixels of an EM-CCD chip than that of the developed system. The developed system can thus detect 1.5-fold more SM spots with the same-size EM-CCD chip, so it can achieve 1.5-fold higher throughput. Moreover, the developed system is based on a simple and practical approach, namely, replacing an ordinary dichroic mirror in a commercially available dual-view optics with a WR DM. This replacement transforms a dual-view imaging system for two-color detection into a system for four-color detection. The developed system is suitable for detection systems of next-generation DNA sequencers and DNA microarray-chip analyzers.     

A fluorescence detection scheme for capillary electrophoresis of N-methylcarbamates with on-column thermal decomposition and derivatization

This paper describes a fluorescence detection method for N-methylcarbamate (NMC) pesticides in micellar electrokinetic chromatography (MEKC) separation. Fulfillment of the fluorescence detection hinged on the discovery that quaternary ammonium surfactants (particularly cetyltrimethylammonium bromide, CTAB), besides serving as hydrophobic pseudophases in MEKC, are also capable of catalyzing the thermal decomposition of NMCs to liberate methylamine. Thus, a multifunctional MEKC medium consisting of borate buffer, CTAB, and derivatizing components (o-phthaldialdehyde/2-mercaptoethanol) was formulated, which allowed first normal MEKC separation, subsequent thermal decomposition, and finally in situ derivatization of NMCs. With careful optimization of the operation conditions, fluorescence detection of 10 NMC compounds was achieved, with column efficiencies typically higher than 50,000 and detection limits better than 0.5 ppm. The present work represents an unprecedented effort in capillary electrophoresis (CE), in which an intact capillary was consecutively utilized as chambers for separation, decomposition, derivatization, and detection, without involving any interfacing features. The success in the implementation of such a detection system resulted in strikingly simple instrumentation as compared with the traditional postcolumn fluorescence determination of NMCs by reversed-phase HPLC. Similar protocols should be workable in the determination of a wide range of pesticides and pharmaceuticals in CE formats.     

Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection

An integrated plastic microfluidic device was designed and fabricated for bacterial detection and identification. The device, made from poly(cyclic olefin) with integrated graphite ink electrodes and photopatterned gel domains, accomplishes DNA amplification, microfluidic valving, sample injection, on-column labeling, and separation. Polymerase chain reaction (PCR) is conducted in a channel reactor containing a volume as small as 29 nL; thermal cycling utilizes screen-printed graphite ink resistors. In situ gel polymerization was employed to form local microfluidic valves that minimize convective flow of the PCR mixture into other regions. After PCR, amplicons (products) are electrokinetically injected through the gel valve, followed by on-chip electrophoretic separation. An intercalating dye is admixed to label the amplicons; they are detected using laser-induced fluorescence. Two model bacteria, Escherichia coli O157 and Salmonella typhimurium, were chosen to demonstrate bacterial detection and identification based on amplification of several of their unique DNA sequences. The limit of detection is about six copies of target DNA.     

Nucleic acid amplification of individual molecules in a microfluidic device

A microfluidic device was developed that enabled rapid polymerase chain reaction (PCR) analysis of individual DNA molecules. The device combined a means for accessing samples serially from a microtiter plate, channels for assembling eight parallel PCR reactions, and integrated resistive heaters for rapid thermocycling (>5 degrees C/s heating, >7 degrees C/s cooling) of samples as they flowed continuously through PCR channels. Amplification was monitored by fluorescence detection of Taqman probes. The long, narrow channels (10 microm x 180 microm x 40 mm) allowed sufficient separation between neighboring DNA templates to enable amplification of discreet DNA molecules. The functionality of the device was demonstrated by reproducibly amplifying a 2D6.6 CYP450 template and distinguishing between wild-type and mutant sequences using Taqman probes. A comparison of the rate of individual amplification events to the expected Poisson distribution confirmed that the device could reliably analyze individual DNA molecules. This work establishes the feasibility of rapid, single-molecule interrogation of nucleic acids.     

Automated and integrated system for high-throughput DNA genotyping directly from blood

An automated and integrated system for DNA typing directly from blood samples has been developed. The multiplexed eight-array system is based on capillary microfluidics and capillary array electrophoresis. Three short-tandem-repeat loci, vWA, THO1, and TPOX, are coamplified simultaneously in a fused-silica capillary by a hot-air thermocycler. Blood is directly used as the sample for polymerase chain reaction (PCR) without any pretreatment. Modifications of standard protocols are necessary for direct PCR from blood. A programmable syringe pump plus a set of multiplexed liquid nitrogen freeze/thaw switching valves are employed for liquid handling in the fluid distribution network. The system fully integrates sample loading, PCR, addition of an absolute standard, on-line injection of sample and standards, separation and detection. The genotypes from blood samples can be clearly identified in eight parallel channels when the electropherograms are compared with that of the standard allelic ladder by itself. Regeneration and cleaning of the entire system prior to subsequent runs are also integrated into the instrument. The instrumentation is compatible with future expansion to hundreds of capillaries to achieve even higher throughput.     

Using commercially available personal glucose meters for portable quantification of DNA

DNA detection is commonly used in molecular biology, pathogen analysis, genetic disorder diagnosis, and forensic tests. While traditional methods for DNA detection such as polymerase chain reaction (PCR) and DNA microarrays have been well developed, they require sophisticated equipment and operations, and thus it is still challenging to develop a portable and quantitative DNA detection method for the public use at home or in the field. Although many other techniques and devices have been reported to make the DNA detection simple and portable, very few of them are currently accessible to the public for quantitative DNA detection because of either the requirement of laboratory-based instrument or lack of quantitative detection. Herein we report application of personal glucose meters (PGMs), which are widely available, low cost, and simple to use, for quantitative detection of DNA, including a hepatitis B virus DNA fragment. The quantification is based on target-dependent binding of cDNA-invertase conjugate with the analyte DNA, thereby transforming the concentration of DNA in the sample into glucose through invertase-catalyzed hydrolysis of sucrose. Instead of amplifying DNA strands through PCR, which is vulnerable to contaminations commonly encountered for home and field usage, we demonstrate here signal amplifications based on enzymatic turnovers, making it possible to detect 40 pM DNA using PGM that can detect glucose only at the mM level. The method also shows excellent selectivity toward single nucleotide mismatches.     

Portable microcoil NMR detection coupled to capillary electrophoresis

High-efficiency separation techniques, such as capillary electrophoresis (CE), coupled to a nondestructive nuclear magnetic resonance (NMR) spectrometer offer the ability to separate, chemically identify, and provide structural information on analytes in small sample volumes. Previous CE-NMR coupled systems utilized laboratory-scale NMR magnets and spectrometers, which require very long separation capillaries. New technological developments in electronics have reduced the size of the NMR system, and small 1-2 T permanent magnets provide the possibilities of a truly portable NMR. The microcoils used in portable and laboratory-scale NMR may offer the advantage of improved mass sensitivity because the limit of detection (LOD) is proportional to the coil diameter. In this work, CE is coupled with a portable, briefcase-sized NMR system that incorporates a microcoil probe and a 1.8 T permanent magnet to measure (19)F NMR spectra. Separations of fluorinated molecules are demonstrated with stopped- and continuous-flow NMR detection. The results demonstrate that coupling CE to a portable NMR instrument is feasible and can provide a low-cost method to obtain structural information on microliter samples. An LOD of 31.8 nmol for perfluorotributylamine with a resolution of 4 ppm has been achieved with this system.     

Detection of 100 aM fluorophores using a high-sensitivity on-chip CE system and transient isotachophoresis

We present a highly sensitive capillary electrophoresis (CE) assay that combines transient, single-interface on-chip isotachophoresis (ITP) and a laser-induced confocal fluorescence detection setup. We performed experimental parametric studies to show the effects of microscope objective specifications and intensity of excitation laser on optimization of a high-sensitivity on-chip CE detection system. Using the optimized detection system, single-molecule detection of Alexa Fluor 488 was demonstrated, and signal data were validated with autocorrelation analysis. We also demonstrated a separation and detection of 100 aM fluorophores (Alexa Fluor 488 and bodipy) in a fast assay using a high-sensitivity on-chip CE detection system and an ITP/CE protocol with no manual buffer exchange steps. This is, to the knowledge of the authors, the highest electrophoretic separation sensitivity ever reported.     

Simple multiplex genotyping by surface-enhanced resonance Raman scattering

The accurate detection of DNA sequences is essential for a variety of post human genome projects including detection of specific gene variants for medical diagnostics and pharmacogenomics. A specific DNA sequence detection assay based on surface-enhanced resonance Raman scattering (SERRS) and an amplification refractory mutation system (ARMS) is reported. Initially, generation of PCR products was achieved by using specifically designed allele-specific SERRS active primers. Detection by SERRS of the PCR products confirmed the presence of the sequence tested for by the allele-specific oligonucleotides. This lead directly to the multiplex genotyping of human DNA samples for the deltaF508 mutational status of the cystic fibrosis transmembrane conductance regulator gene using SERRS active primers in an ARMS assay. Removal of the unincorporated primers allowed fast and accurate analysis of the three genotypes possible in this system in a multiplex format without any separation of amplicons. The results indicate that SERRS can be used in modern genetic analysis and offers an opportunity for the development of novel assays. This is the first demonstration of the use of SERRS in multiplex genotyping and shows potential advantages over fluorescence as a detection technique with considerable promise for future development.     

Fluorescence lifetime for on-the-fly multiplex detection of DNA restriction fragments in capillary electrophoresis

This paper describes the first use of frequency-domain fluorescence lifetime for multiplex detection of DNA restriction fragments in capillary electrophoresis (CE). The fragments were labeled with monomeric intercalating dyes that can be excited by either the 488- or 514-nm line of an argon ion laser and have lifetimes in the range of 0.5-2.5 ns. We were able to achieve multiplex lifetime detection in the CE separation of a restriction fragment digest and a DNA size ladder in the same run, for fragments shorter than 700 bp. Different gel buffer systems, including a modified polyacrylamide gel and several tris-borate-EDTA/hydroxyethylcellulose (TBE/HEC) gels, were investigated for separation and detection of the dye-labeled DNA fragments. Best results for both electrophoretic resolution and lifetime detection were obtained using a gel containing 1% high molecular weight (90,000-105,000) HEC and 0.3% low molecular weight (24,000-27,000) HEC in TBE buffer.     

Single-molecule DNA amplification and analysis in an integrated microfluidic device

Stochastic PCR amplification of single DNA template molecules followed by capillary electrophoretic (CE) analysis of the products is demonstrated in an integrated microfluidic device. The microdevice consists of submicroliter PCR chambers etched into a glass substrate that are directly connected to a microfabricated CE system. Valves and hydrophobic vents provide controlled and sensorless loading of the 280-nL PCR chambers; the low volume reactor, the low thermal mass, and the use of thin-film heaters permit cycle times as fast as 30 s. The amplified product, labeled with an intercalating fluorescent dye, is directly injected into the gel-filled capillary channel for electrophoretic analysis. Repetitive PCR analyses at the single DNA template molecule level exhibit quantized product peak areas; a histogram of the normalized peak areas reveals clusters of events caused by 0, 1, 2, and 3 viable template copies in the reactor and these event clusters are shown to fit a Poisson distribution. This device demonstrates the most sensitive PCR possible in a microfabricated device. The detection of single DNA molecules will also facilitate single-cell and single-molecule studies to expose the genetic variation underlying ensemble sequence and expression averages.