High-throughput genotyping of repeat polymorphism in the regulatory region of serotonin transporter gene by gel microchip electrophoresis.

Large-scale genotyping of the repeat polymorphism in the regulatory region of the serotonin transporter gene (5-HTTLPR) was attempted by polymerase chain reaction (PCR) amplification followed by gel microchip electrophoresis analysis. The multilane (96) format of the gel microchip system allowed parallel separation of a large number of samples. The separation and visualization of the PCR amplicons from either the 5-HTTLPR short allele (number of repeats are 14) or the 5-HTTLPR long form (16 repeats) was completed in a few minutes. Genotyping of healthy Caucasian individuals showed that the short allele had a somewhat lower frequency (0.42) than the long form (0.58), and the genotype frequencies fulfilled the criteria of the Hardy-Weinberg equilibrium (chi = 0.012, p = 0.994). Based on these results, gel microchip electrophoresis system proved to be a powerful tool for high throughput genotyping of repeat polymorphism.     

Very fast capillary electrophoresis with electrochemical detection for high-throughput analysis using short,vertically aligned capillaries

A method for conducting fast and efficient capillary electrophoresis (CE) based on short separation capillaries in vertical alignment was developed. The strategy enables for high-throughput analysis from small sample vials (low microliter to nanoliter range). The system consists of a lab-made miniaturized autosampling unit and an amperometric end-column detection (AD) cell. The device enables a throughput of up to 200 separations per hour. CE-AD separations of a dye model system in capillaries of only 4 to 7.5 cm length with inner diameters (ID) of 10 or 15 μm were carried out under conditions of very high electric field strengths (up to 3.0 kV/cm) with high separation efficiency (half peak widths below 0.2 s) in less than 3.5 s migration time. A non-aqueous background electrolyte, consisting of 10 mM ammonium acetate and 1 M acetic acid in acetonitrile, was used. The practical suitability of the system was evaluated by applying it to the determination of dyes in overhead projector pens. Fig. 1

Schematic illustration of high-throughput capillary electrophoresis with electrochemical detection     

Characterization of etched electrochemical detection for electrophoresis in micron inner diameter capillaries

An enhanced etched electrochemical (EC) detection technique has been developed for CE in micron inner diameter capillaries. The design improvements allow for better alignment between the capillary bore and the electrode. This new method involves utilizing a carbon fiber microelectrode and etching both the carbon fiber and the detection end of a micrometer-sized inner diameter capillary to limit dead volume and analyte diffusion at the amperometric EC detector. To understand the factors affecting enhanced detector efficiency, a detailed examination of the relationship between detector design and performance has been completed by exploring the effects of varying electrode diameter, tip shape, and size, in addition to the etch length of the capillary outlet. The enhanced detection provides peak efficiencies as high as 75000 theoretical plates and estimated detection limits as low as 40 nM for dopamine. This etched detection method should further facilitate volume-limited sample analysis by CE.     

Fast electrophoresis in conventional capillaries by employing a rapid injection device and contactless conductivity detection

A purpose-made set-up featuring an automated fast injector allowed the easy optimization of the injected amount and the adjustment of the separation length of conventional capillaries from a minimum of 5 cm upward. It was found that a compromise in capillary length for separation efficiency and analysis time also has to take into account the injected amount, which in turn affects the sensitivity and hence the detection limit. The versatility of the system was demonstrated by the analysis of the major cations and anions in natural water samples in less than 1 min, the concurrent determination of a mixture of amino acids and carbohydrates in 160 s, and of three active ingredients in a pharmaceutical preparation in 40 s. Plate numbers were typically around 50,000 and detection limits down to 1 M could be achieved.     

Sizing short tandem repeat alleles in capillary array gel electrophoresis instruments.

A 96-capillary array gel electrophoresis Applied Biosystems 3700 instrument has been used to analyse AMPF/STR SGM Plus short tandem repeat (STR) loci for forensic applications. This multiplex consists of ten STR loci plus the Amelogenin locus and currently forms the basis of the UK National DNA database that currently holds more than 1 million profiles. Of particular interest is the accuracy of allele designation that is determined by comparison with standard control allelic ladder markers. Some loci have higher standard deviations than others. In particular the high-molecular-weight HUMFIBRA alleles have high standard deviations of the order of 0.15 and it is these alleles that are most likely to be misdesignated. However, this risk is minimised by the analysis of at least five different allelic ladders across the array to estimate the mean size of each allele. In conjunction with this, a series of guidelines that can be programmed into expert systems are used to minimise risks of misdesignation. The efficacy of the procedures utilised are tested by computer simulation and demonstrated to be robust.     

A single-strand conformation polymorphism method by capillary electrophoresis with laser-induced fluorescence for detection of the T1151A mutation in hMLH1 gene

Mutation of hMLH1 gene plays an important role in human tumorigenesis. A highly sensitive single-strand conformation polymorphism (SSCP) method for detection of the T1151A mutation in exon 12 of the hMLH1 gene was for the first time developed employing laser-induced fluorescence capillary electrophoresis (LIF-CE). Effects of the concentration of linear polyacrylamide solution, running temperature, running voltage and the addition of glycerol on SSCP analysis were investigated, and the optimum separation conditions were defined. Thirty colorectal cancer patients and eight lung cancer patients were screened and the T1151A mutation was found in four of them. Based on CE-sequencing the mutation was further confirmed. To our knowledge, this is for the first time that the T1151A mutation is found in lung cancer. Our method is simple, rapid, and highly sensitive and is well suited to the analysis of large numbers of clinical samples.     

Design and evaluation of capillary electrophoresis in dynamically coated capillaries coupled with chemiluminescence detection

A dynamic coating capillary electrophoresis coupled with a simplified on-line chemiluminescence detection system was designed and evaluated. In the proposed system, poly-vinylpyrrolidone was used as dynamic coating substance in the separation buffer to reduce the unwanted protein non-specific adsorption, which was first applied in capillary electrophoresis coupling with on-line chemiluminescence detection. In order to avoid complex processing, an ordinary plastic cuvette was modified as a three-way joint. The chemiluminescence reaction conditions and capillary electrophoresis separation conditions were investigated in detail. The results showed that the coated capillary can be injected protein samples at least 30 times continuously with good repeatability. Under optimal conditions, the chemiluminescence relative intensity was linear with the concentration of hemoglobin in the range of 4-1850 μg mL(-1) and the detection limit was 2.0 μg mL(-1) (S/N=3). The relative standard deviation of migration times and peak heights for 40 μg mL(-1) hemoglobin were 2.5% and 4.1% (n=11) respectively. Interference of matrix effects was overcome by the calibration according to standard addition methods. Afterwards, the method was validated successfully and was applied to detect the concentration of hemoglobin in the serum of haemolytic patients.     

Optimum sample medium for single-nucleotide polymorphism and mutation detection by capillary electrophoresis

Capillary electrophoresis (CE) is a versatile analytical platform widely used for nucleic acids analysis. Its applications in research and diagnostics include scanning and screening for mutations and polymorphisms by such reliable methods as single-strand conformation polymorphism (SSCP), heteroduplex analysis (HA), and combined SSCP/HA. This study, aimed at the further development of these methods, is focused on detailed sample-media characteristics. Factors affecting single-strand conformer stability and DNA intake efficiency were analyzed. The sample media optimal for efficient mutation or SNP detection were determined, and complex SSCP-CE patterns arising from unpurified PCR products were explained. It turns out, that the nondenaturing aqueous media assure both efficient DNA intake, and single-strand conformers stability required for the SSCP and combined SSCP/HA. The results of this study are applicable to all these areas of biomedical research, in which capillary electrophoresis is used for the characterization of nucleic acids.     

Validation of the HumDXS6807 short tandem repeat polymorphism for forensic application.

This paper presents sequence and population genetic data of the HumDXS6807 short tandem repeat (also known as CHLC.GATA52B03) which is a tetranucleotide repeat polymorphism representing seven alleles of 251-275 bp in length. HumDXS6807 is located at Xpter-Xp22.2, i.e., at a genetic distance of more than 87 and 151 cM from the well-known markers HumARA and HumHPRTB, respectively. Kinship tests in 157 family trios revealed a typical X-linked codominant inheritance; mutations were not found. Population genetic data were obtained by analyzing a Caucasian population sample comprising 308 females and 209 males: polymorphism information content (PIC) = 0.640; Heterozygosity (Het) = 0.668; Mean exclusion chance (MEC) = 0.414. The HumDXS6807 allele distribution met the Hardy-Weinberg expectations.     

Temperature and pH studies of short tandem repeat systems using capillary electrophoresis at elevated pH

The DNA secondary structure can affect the migration time and precision of DNA separations in the physical gels used in capillary electrophoresis (CE). To counteract these effects, DNA typing is performed using elevated temperatures (60 degrees C) and high concentrations (7 M) of urea. These conditions affect the precision and lifetime of the analysis. To better understand the effects of these conditions on the reproducibility of DNA migration, we examined the effects of temperature and pH on short tandem repeat (STR) analysis using the PE/ABI 310 Genetic Analyzer. Separations were performed using the Profiler + multiplex system, a set of coamplified STRs with a 4-base repeat motif, labeled at the 5'-end using fluorescent dyes. The analytical separations were obtained using a commercial buffer at pH 8 and an experimental buffer consisting of 3% hydroxyethylcellulose at pH settings ranging from 8-12. Multichannel laser-induced fluorescence detection was used. Temperatures were examined from 30-70 degrees C. The results demonstrate the fact that highly efficient separations can be carried out at alkaline pH. In addition, improvements in temperature stability were seen when compared to results at lower pH. However, high concentrations of urea were found to be necessary to achieve optimal resolution.     

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.     

Determination of sulfate and chloride ions in highly saline oilfield water by capillary electrophoresis using bilayer-coated capillaries and indirect absorption detection

Analysis of highly saline oilfield waters for anions presents challenges. Traditional analytical techniques used for such analysis tend to suffer from both poor sensitivity and selectivity due to the high concentrations of salt present in the samples. A capillary electrophoresis method was developed for the simultaneous determination of chloride and sulfate anions which is relevant to the oilfield analysis industry and of economic value. Due to the extremely high concentrations of chloride in highly saline oilfield waters, it is difficult to achieve baseline electrophoretic separation necessary for accurate quantitation. By using a capillary with a noncovalently bound bilayer coating using Polybrene, a cationic polymer and sodium dodecyl sulfate (SDS), an anionic surfactant and a buffer consisting of 50 mM TRIS, 30 mM SDS, 5% methanol and 26 mM chromium trioxide (CrO₃) at pH 6.7, baseline separation (R_s > 1.5) of chloride and sulfate was achieved. To mimic possible oilfield water samples, model water solutions of 5%, 10%, 15% and 20% chloride containing low ppm sulfate were prepared and successfully analysed using the method developed. In addition, the method was applied to determine chloride and sulfate anions in highly saline oilfield water samples. The accuracy of the method developed was verified by analysing NIST certified standards of chloride and sulfate. The results obtained for chloride and sulfate with the indirect CE-UV method were in close agreement (94–100% accuracy; <2.5% relative standard deviations) with those of the certified standard analysed by ion chromatography.     

Detection of errors in dinucleotide repeat typing by nondenaturing electrophoresis.

This study shows that consideration of minor bands (heteroduplex, shadow, and faint bands) associated with allele bands in nondenaturing polyacrylamide gel electrophoresis (PAGE) after polymerase chain reaction (PCR) is effective for detecting PCR processing errors that lead to mistyping of heterozygotes as homozygotes. Notably, we show that minor bands in native gels are highly effective for detecting allele dropout and preferential amplification in PCR amplification of dinucleotide repeats. These findings are based on an analysis of Mendelian inheritance patterns in families, and the reproduction of heterozygous band patterns by mixing homozygous DNAs before PCR, for a total of six (AC)n repeats located on human chromosome 11p15. To investigate the utility of our approach, a large population sample of 405 unrelated individuals was genotyped for each (AC)n repeat using minor bands as internal quality controls. Genotype frequencies at each of the six loci were in close agreement with Hardy-Weinberg proportions, which suggests that there were few genotyping errors. Our observations add to the evidence indicating that minor bands in native gels are of diagnostic value in the genotyping of dinucleotide repeats.     

Validation of a tentative microsatellite marker for the dopamine D4 receptor gene by capillary gel electrophoresis

Two to four-basepair-short tandem repeats (i.e. microsatellites) are broadly utilized as genetic markers for mapping disease loci in whole genome search analyses. Based on their close vicinity on chromosome 11, the D11S1984 microsatellite was anticipated as a tentative marker for the dopamine D4 receptor gene. A capillary gel electrophoresis based genotype analysis method and an in-house made computational tool was developed for the analysis of the D11S1984 microsatellite marker to examine a healthy Hungarian population of n=106. The data obtained did not suggest significant linkage between the D11S1984 marker and the DRD4 gene.     

Analysis of dopamine D4 receptor gene polymorphism using microchip electrophoresis

A microfabricated electrophoresis device was used for rapid polymerase chain reaction product analysis in genotyping the dopamine D4 receptor gene (DRD4) 48 base pairs repeat polymorphism. An allelic ladder, prepared from homozygous individuals, was used as internal standard during the microchip electrophoresis based analysis. Comparison of this novel separation method with the conventional slab gel and previously reported ultra-thin-layer techniques confirmed the reliability of this new method. Genotyping of 332 healthy Hungarian individuals gave the following allele frequencies: two-repeat: 0.089; three-repeat: 0.026; four-repeat: 0.674; five-repeat: 0.011; six-repeat: 0.002; seven-repeat: 0.189; eight-repeat: 0.011. The genotype frequencies obtained showed no deviation from the Hardy-Weinberg equilibrium (p>0.903), further underlying the reliability of this new genotyping technique.     

Characterization of a highly variable short tandem repeat polymorphism at the D2S1242 locus

We report the evaluation of short tandem repeat (STR) locus D2S1242 (GDB ID G00-309-429) for forensic purposes, investigated by polymerase chain reaction (PCR) amplification and both native and denaturating polyacrylamide gel electrophoresis in 147 unrelated Austrians. No deviations from Hardy-Weinberg expectations were observed. The mean exclusion chance (MEC) was 0.669, the discriminating power (DP) was 0.947, and the observed heterozygosity rate was 0.856. An allelic ladder consisting of eight sequenced alleles (141-167 and 175 bp) was constructed. Sequence analysis revealed that the locus comprised two repeat motifs varying in number between alleles GAAA and GAAG. According to the number of tetranucleotide repeats the smallest allele was designated as 10 and the largest allele as 18.     

Some important combinations of detection techniques for electrophoresis in capillaries and on chips with emphasis on electrochemical principles

Analyses of very complex samples involving capillary and chip electrophoresis often require dual (multiple) detection to attain sufficient selectivity of determination. The present work reviews and critically evaluates selected combinations of electrochemical detection techniques among themselves and with absorption spectrometric, fluorescence and luminescence techniques. Amperometry, contact and contactless conductometry, UV photometry and fluorescence measurements are paid special attention. Some information is also given on combinations of spectrometric techniques with mass spectrometry. The properties of the detection systems are critically discussed, examples are illustrated in figures and some details on the characteristics of dual detectors and their applications are tabulated.     

Direct determination of bromide ions in seawater by capillary zone electrophoresis using polyethyleneimine-coated capillaries

A rapid and simple capillary electrophoretic method was developed for the direct determination of bromide ion in seawater. We have found an effective method, based on the use of polyethyleneimine-coated capillaries and the addition of sodium chloride to the background electrolyte. The use of coated capillaries with a cationic polymer changes the direction of the electroosmotic flow in the capillary, which favors the migration speed of the bromide ion and enables the use of low salt concentrations in the separation electrolyte. Bromide ion in seawater can be determined within 2 min using this system and 20 mmol L^-1 NaCl-containing separation electrolyte. The detection limit for the bromide ion was 0.45 g ml^-1. The method was applied to the determination of bromide ion in seawater samples collected from the Bosphorus and the Black Sea. Bromide contents in samples from 0 to 72 m depths varied between 33.2 and 72.8 mg L^-1 with a mean 3.0% RSD.     

Evaluation and validation of the ABI 3700, ABI 3100, and the MegaBACE 1000 capillary array electrophoresis instruments for use with short tandem repeat microsatellite typing in a forensic environment

The demand for high-throughput DNA profiling has increased with the introduction of national DNA databases and has led to the development of automated methods of short tandem repeat (STR) profile production; however, a potential bottleneck still exists at the gel electrophoresis stage. Capillary electrophoresis sequencers capable of processing 96 samples with considerably reduced manual intervention are now available. In this paper, we compare the ABI Prism 377 slab-gel sequencer currently used by the Forensic Science Service with three leading capillary array electrophoresis instruments: the ABI Prism 3700, the Amersham MegaBACE 1000 and the 16-capillary ABI Prism 3100. We describe the experiments used to evaluate and validate these platforms for forensic use with the STR multiplex Ampf/STR SGMplus [1, 2], along with comparative data from the ABI Prism 377 sequencer.