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.

Functional analysis and treatment of self-injury associated with transitions.

We applied functional analysis methodology to the assessment and treatment of 2 individuals' self-injurious behavior (SIB), which was reported to be occasioned by transitions from one activity or location to another. A structural (task) analysis of activity transitions identified at least three separate components that might influence behavior either alone or in combination: (a) termination of a prechange activity, (b) initiation of a postchange activity, and (c) movement from one location to another. Results of preference and avoidance assessments were used to identify activities to which participants were exposed in varying arrangements during transitions in a functional analysis. Results of 1 participant's functional analysis indicated that his SIB was maintained by avoidance of having to change locations, regardless of the activity terminated prior to the change or the activity initiated following it. The 2nd participant's analysis revealed the same function but also an additional one: avoidance of certain task initiations. This information was used to identify transition contexts during intervention and to design treatment procedures appropriate for a given context and behavioral function. A procedure involving advance notice of an upcoming transition had no effect on SIB, and differential reinforcement of alternative behavior (DRA) had limited effects in the absence of extinction. Sustained decreases in SIB were observed when DRA was combined with extinction and response blocking. Further extensions of functional analysis methodology to the assessment of problem behavior in situations characterized by multiple or protracted stimulus changes are discussed.

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.

Functional analysis and treatment of problem behavior evoked by noise.

We conducted a four-part investigation to develop methods for assessing and treating problem behavior evoked by noise. In Phase 1, 7 participants with developmental disabilities who were described as being hypersensitive to specific noises were exposed to a series of noises under controlled conditions. Results for 2 of the participants verified that noise was apparently an aversive event. In Phase 2, results of functional analyses indicated that these 2 participants' problem behaviors were maintained by escape from noise. In Phase 3, preference assessments were conducted to identify reinforcers that might be used during treatment. Finally, in Phase 4, the 2 participants' problem behaviors were successfully treated with extinction, stimulus fading, and a differential-reinforcement-of-other-behavior (DRO) contingency (only 1 participant required DRO). Treatment effects for both participants generalized to their home environments and were maintained during a follow-up assessment. Procedures and results were discussed in terms of their relevance to the systematic assessment of noise as an establishing operation (EO) and, more generally, to the identification of idiosyncratic EO influences on behavior.

Advances ofcapillary electrophoresis in clinical and forensic analysis (1999-2000).

In this paper, capillary electrophoresis in clinical and forensic analysis is reviewed on the basis of the literature of 1999, 2000 and the first papers in 2001. An overview of progress relevant examples for each major field of application, namely (i) analysis of drug seizures, explosives residues, gunshot residues and inks, (ii) monitoring of drugs, endogenous small molecules and ions in biofluids and tissues, (iii) general screening for serum proteins and analysis of specific proteins (carbohydrate deficient transferrin, alpha1-antitrypsin, lipoproteins and hemoglobins) in biological fluids, and (iv) analysis of nucleic acids and oligonucleotides in biological samples, including oligonucleotide therapeutics, are presented.

Application of a low cost telemedicine link to the diagnosis of neonatal congenital heart defects by remote consultation.

OBJECTIVE: To determine whether accurate remote echocardiographic diagnosis of congenital heart disease could be achieved using a low cost telemedicine system. DESIGN: Echocardiographic images obtained by a paediatrician from neonates suspected of having congenital heart disease were transmitted by a telemedicine link across two integrated service digital network (ISDN) lines to a regional paediatric cardiology unit for interpretation by a consultant paediatric cardiologist. The "tele-echo" diagnosis was verified by the paediatric cardiologist on direct consultation and echocardiography. SETTING: Neonatal unit of Altnagelvin Hospital, Londonderry (a district general hospital) and the regional paediatric cardiology department, Royal Belfast Hospital for Sick Children. MAIN OUTCOME MEASURES: Accuracy of the diagnosis made using the telemedicine link; impact on patient management. RESULTS: Between September 1995 and September 1997 echocardiographic images were transmitted on 63 patients. A diagnosis was made in 61 (97%) (transmitted images were unsatisfactory in two). Congenital heart disease was diagnosed in 42 patients. Fourteen patients with major congenital heart disease were accurately diagnosed within 24 hours of admission using the telemedicine link and were transferred to the regional paediatric cardiology unit. A further 28 with less serious congenital heart disease continued to be managed at the district general hospital. Congenital heart disease was excluded in 19. Follow up consultation confirmed accurate diagnosis or exclusion of congenital heart disease in 57 (93%). There were four inaccurate diagnoses (6.3%; three undetected small ventricular septal defects and one pulmonary stenosis). CONCLUSIONS: Transmitted images were of sufficient quality to allow confirmation or exclusion of major congenital heart disease. The telemedicine link facilitated early diagnosis and initiation of appropriate management in patients with complex congenital heart disease and avoided the need for transfer in those where significant congenital heart disease was excluded.

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.

Value of a low-cost telemedicine link in the remote echocardiographic diagnosis of congenital heart defects.

We established a low-cost telemedicine link from a district general hospital to the regional paediatric cardiology department about 120 km away. The link was used to transmit echocardiographic images of newborn infants suspected of having congenital heart disease (CHD) to the referral centre, with simultaneous video and audio contact for consultation. Echocardiograms were transmitted for 61 patients suspected of having CHD, aged from 1 to 42 days. The transmitted images were of adequate quality for the paediatric cardiologist to make a diagnosis in 59 (97%). Congenital heart abnormalities were diagnosed in 38 (64%). Twelve patients (20%) had major CHD diagnosed on the transmitted scan and required transfer to the regional cardiology unit either urgently or electively after initial measures to stabilize the patient. Our findings suggest that, for babies suspected of having CHD, ultrasound images of diagnostic quality can be obtained and transmitted using a low-cost telemedicine system.

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.

Long term renal prognosis of Henoch-Schönlein purpura in an unselected childhood population.

All 270 patients presenting with Henoch-Schönlein Purpura over a 13-year period from a total childhood population of 155,000 were studied. This is an incidence of 13.5/100,000 children per year. Fifty-five (20%) were found to have initial evidence of renal involvement, and were re-examined at a mean of 8.3 years later. The 37 with isolated haematuria or haematuria with mild proteinuria (67%) recovered completely. Eighteen (33%) had nephritic or combined nephritic/nephrotic features, of these one died in the acute phase of the illness, only three others have persistent urinary abnormalities but have no biochemical evidence of renal impairment and only minor histological changes. The overall prognosis in this unselected population is therefore good with a mortality less than 1% overall and low long term morbidity of 1.1%. This study indicates a more optimistic outcome in this condition than the majority of published estimates based on more selected groups of patients.