Nonradiochemical DNase I footprinting by capillary electrophoresis.

A new application for DNase I footprinting using capillary electrophoresis (CE) has been developed in order to decrease analysis time and to eliminate the use of radiochemicals. An additional advantage of the new method over the traditional radioactive methods is that the DNA probe can be labeled on both ends with different fluorescein dyes. This provides an internal check of the identification of protein-binding sites on DNA, because the binding region can be observed from both DNA strands. The initial parameters for the CE method were developed using the Promega Core Footprinting Kit for analysis of AP-2 binding sites in the SV40 enhancer sequence. After optimization of the method, the protocol was found to be effective for footprint analysis of the immediate upstream region (bases -1 to -370) of the rat glutathione peroxidase (GPX) and it permitted identification of a previously unknown binding site in the upstream sequence of the GPX gene.

Novel electrolyte for the analysis of cations in low explosive residue by capillary electrophoresis.

A novel electrolyte has been developed for the effective separation by capillary electrophoresis of cations detected in low explosive residue. This electrolyte, with a pH of 4.4, employs 17.5 mM alpha-hydroxyisobutyric acid (HIBA) as the complexing agent, 6 mM imidazole as the ultraviolet visualization agent, 4 mM 18-crown-6 ether as a modifier to enhance the selectivity of the inorganic cations, and 5% (v/v) acetonitrile as an organic additive. Studies which assessed the value of the addition of 18-crown-6 and acetonitrile demonstrated conclusively that both were required in order to achieve unambiguous baseline separation of ammonium, potassium and monomethylammonium ions. The major advantages of the use of this electrolyte are a total run time of less than 7 min and symmetrical peak shapes. Validation on a series of preblast and postblast explosives materials determined that this procedure is reliable and robust.

Analysis of two multiplexed short tandem repeat systems using capillary electrophoresis with multiwavelength fluorescence detection.

A series of experiments was performed to analyze the utility of capillary electrophoresis (CE) with multiwavelength detection capabilities for multiplex typing of short tandem repeat loci. Characteristics of the sieving polymer, hydroxyethylcellulose, which affect resolution of single strand (ss) DNA fragments were examined. Additionally, the effects of denaturant in the polymer system, separation voltage, and analysis temperature were studied to ascertain their effects on DNA separations and capillary lifetime. The use of elevated run temperature (60 degrees C) was found to improve sizing precision, to increase the lifetime of capillaries (100 runs or more per capillary), and to provide runtimes of under 20 min. Finally, 100 individual human DNA samples were typed successfully using CE. The average resolution obtained was 1.4 bases for a 200 base fragment with a standard deviation of sizing of 0.2 bases, allowing all alleles examined to be distinguished clearly.

Analysis of multiplexed short tandem repeat (STR) systems using capillary array electrophoresis.

The profiling of polymorphic short tandem repeat (STR) markers is being applied to human identification, parentage testing and genetic mapping. Reliable genotyping of these markers is facilitated by polymerase chain reaction (PCR) amplification and high-resolution electrophoretic separation. Capillary array electrophoresis (CAE) offers very rapid, high-resolution separation of the amplified DNA and potential for automated sample processing not realized employing conventional slab-gel electrophoresis. The use of CAE to type DNA samples amplified at 11 genetic loci in multiplex profiles is presented. Two sets totaling 208 samples were amplified in a multiplex fashion using AmpFlSTR-Blue or AmpFlSTR-Green I and analyzed in a blind study using CAE. With the exception of one sample, the CAE genotyping results were in complete agreement with results obtained using a single-capillary system or two slab-gel electrophoresis systems. The sample, genotype TH01 7/10, migrated similar to TH01 6.3/9.3 allele sizes, which suggested a potential band migration shift. The recommended approach to such an observation is to analyze the sample again. The sample was rerun and correct genotype verified. Allelic ladder samples were analyzed multiple times by CAE to determine sizing accuracy and precision. The sizing of over 240 allelic ladder samples yielded an average within-run precision of +/- 0.13 bp and between-run precision of +/- 0.21 bp for fragments up to 350 bp. The CAE protocols permit processing of up to 96 multiplex STR samples in under 70 min.

The analysis of EDTA in dried bloodstains by electrospray LC-MS-MS and ion chromatography.

Analytical methods were developed to determine the presence of ethylenediaminetetraacetic acid (EDTA) in dried bloodstains to provide probative information when allegations of evidence tampering have been made in criminal cases. A simple screening method using ion chromatography to analyze stains was found to be quantitative to the 5 ppm level. The presence of EDTA was then confirmed using negative and positive ion mode liquid chromatography-tandem mass spectrometry (LC-MS-MS) methods. A blind trial of these methods on 42 samples correctly determined the bloodstains that did and did not contain the preservative EDTA. One interesting observation in these results was the adsorption and postanalysis release of EDTA in the chromatographic system. In order to avoid cross contamination of samples resulting from this phenomena, it was found to be necessary to use EDTA-free blood extracts as blanks in the LC-MS analysis of bloodstains.

DNA typing of a polymerase chain reaction amplified D1S80/amelogenin multiplex using capillary electrophoresis and a mixed entangled polymer matrix.

In this study, a technique was developed to separate by capillary electrophoresis (CE) the widely varying DNA fragment sizes produced by a multiplex polymerase chain reaction (PCR) amplification of the loci D1S80 and amelogenin. Experiments were performed to analyze different buffer systems and obtain optimal resolution for the separation. A matrix composed of two different molecular weights of the same polymer was constructed to separate the DNA fragments with baseline resolution, and a cubic spline fit was used to estimate the size of DNA fragments over 350 base pairs. Over 100 samples were examined to demonstrate the rapid, robust and precise characteristics of this CE system. An average relative standard deviation of 0.3% was obtained for the sizing of the D1S80 alleles in these samples. DNA from mixed body fluid samples, samples subjected to environmental insult, and D1S80 sequence variants were also typed successfully. These results demonstrate that CE is a viable method for analysis of D1S80 and amelogenin forensic DNA samples.

Application of dual internal standards for precise sizing of polymerase chain reaction products using capillary electrophoresis.

Capillary electrophoresis (CE) is an analytical technique which provides rapid, high resolution analysis of amplified DNA fragments produced by the polymerase chain reaction (PCR). In this study, two internal standards are used as size markers to bracket und precisely size PCR products. The technique is applied to typing PCR products from the short tandem repeat locus HUMTH01. HUMTH01 consists of five to seven major alleles in the size range of 179-203 bp, with each allele four bp apart. Using this genetic marker, a population containing 97 individuals was examined with both polyacrylamide gel electrophoresis and CE. Identical genotypes were obtained with both techniques demonstrating the reliability of CE in DNA typing applications. The DNA analysis took place in sets of 10 with a calibration of the CE being performed between each set of samples. For the 97 samples examined, the pooled standard deviation was 0.3 bp. The observed genotype frequencies determined from the sample set did not deviate significantly from Hardy-Weinberg expectations. From these CE results, we conclude that HUMTH01 PCR products can be accurately and precisely sized by capillary electrophoresis using the method described.

Rapid analysis of the short tandem repeat HUMTH01 by capillary electrophoresis.

Using capillary electrophoresis, we demonstrate separation and analysis of the short tandem repeat HUMTH01 in under 10 min with 3 bp resolution. Separation of the PCR products, which range in size from 179 to 203 bp, is achieved using hydroxyethyl cellulose as the separation medium and a novel single-step voltage gradient. Internal standards on either side of the alleles are used to size the PCR products with an average standard deviation of 0.5 bp. DNA typing patterns obtained with this system are compared to samples separated by polyacrylamide slab gel electrophoresis.

Quantitation of polymerase chain reaction products by capillary electrophoresis using laser fluorescence.

In samples where the amount of DNA is limited, the polymerase chain reaction (PCR) can amplify specific regions of the DNA. A quantitative analysis of the PCR product would be desirable to ensure sufficient DNA is available for analysis. In this study, we examine the use of capillary electrophoresis (CE) with laser fluorescence detection for quantitation of PCR products. A coated open tubular capillary was used with a non-gel sieving buffer and a fluorescent intercalating dye to obtain results within 20 minutes. Using an internal standard, peak migration time was below 0.1% relative standard deviation (R.S.D.) with a peak area precision of 3% R.S.D. In comparison to quantitation by hybridization, (i.e., slot blot) and spectrophotometric analysis, capillary electrophoresis shows distinct advantages due to its ability to separate unincorporated primers and PCR byproducts from the targeted PCR product. The results demonstrate that CE can be used to monitor the quality and quantity of the PCR product.