PubMed Health⌕ Search

Biomedical subjects

E S Winn-Deen

Publications and source records attributed to E S Winn-Deen.

10 recordsLinked to original sources
PubMed1997

Optimization of the performance of the polymerase chain reaction in silicon-based microstructures.

We have demonstrated the ability to perform real-time homogeneous, sequence specific detection of PCR products in silicon microstructures. Optimal design/ processing result in equivalent performance (yield and specificity) for high surface-to-volume silicon structures as compared to larger volume reactions in polypropylene tubes. Amplifications in volumes as small as 0.5 microl and thermal cycling times reduced as much as 5-fold from that of conventional systems have been demonstrated for the microstructures.

Actins↗
PubMed1997

Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.

Alleles↗
PubMed1995

Fluorescence-based oligonucleotide ligation assay for analysis of cystic fibrosis transmembrane conductance regulator gene mutations.

Isolation of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), provided a basis for analyzing its molecular pathology and resulted in the identification of > 400 mutations associated with disease. Except for the delta F508 mutation, no other single mutation accounts for > 5% of CF chromosomes in most populations, and most mutation frequencies are < 1%. A strategy based on multiplex PCR followed by multiplex allele-specific oligonucleotide probe ligation was used to detect 30 mutations, distributed throughout ten exons and seven introns of the CFTR gene, that together account for > 96% of CF mutant chromosomes worldwide. Mutations were detected by competitive oligonucleotide probe ligation to detect normal and/or mutant genotypes in one reaction. Three probes (one common and two allelic probes) were needed for analysis of each mutation. Probes hybridized to target DNA were joined by a thermostable ligase if there were no mismatches at their junctions; temperature cycling resulted in a linear increase in product. Common probes were labeled with fluorochromes, and allelic probes each had different lengths. Ligation products were analyzed electrophoretically on a fluorescent DNA sequencer. The results show that combined PCR and probe ligation amplification rapidly and reliably screen for CF homozygotes and carriers.

Alleles↗
PubMed1994

High-density multiplex detection of nucleic acid sequences: oligonucleotide ligation assay and sequence-coded separation.

We describe a non-isotopic, semi-automated method for large-scale multiplex analysis of nucleic acid sequences, using the cystic fibrosis transmembrane regulator (CFTR) gene as an example. Products of a multiplex oligonucleotide ligation assay (OLA) are resolved electrophoretically from one another and from unligated probes under denaturing conditions with fluorescence detection. One ligation probe for each OLA target carries a fluorescent tag, while the other probe carries an oligomeric non-nucleotide mobility modifier. Each OLA product has a unique electrophoretic mobility determined by the ligated oligonucleotides and the mobility-modifier oligomer arbitrarily assigned (coded) to its target. The mobility range for practical mobility modifiers is much wider than the accessible range from unmodified ligated oligonucleotides of practical length. Each mobility modifier is built from phosphoramidite monomers in a stepwise manner on its associated oligonucleotide using an automated synthesizer. The resulting mobility modifiers lower the probe-target duplex Tm by less than 3 degrees C and retard probe-target annealing by less than 50%, with negligible effect on OLA yield and specificity. This method is especially useful for allelic discrimination in highly polymorphic genes such as CFTR.

Autoanalysis↗
PubMed1993

Ligation amplification and fluorescence detection of Mycobacterium tuberculosis DNA.

Current methods for the identification of Mycobacterium tuberculosis are dependent upon culture of the bacteria and are necessarily lengthy due to the slow growth of this agent. The development of DNA probe technology offers rapid, accurate and cost effective alternatives for the identification of such fastidious organisms. A technique for detecting specific DNA sequences, known as oligonucleotide ligation assay (OLA) involves the ligation of two adjacent oligonucleotides annealed to target DNA, and has been previously described. Amplification of the target sequences can be accomplished by including complementary pairs of oligonucleotides and a thermal stable ligase in a reaction which cycles between annealing/ligation and denaturing temperatures. Using a cloned portion of an insertion sequence, IS6110, which has been reported to be specific for M. tuberculosis complex as target DNA, we demonstrate the ligation dependent amplification of a 40 base pair region of plasmid bearing IS6110. By employing oligonucleotides which are each labelled with a different fluorescent dye, the reaction can be followed by fluorescence detection on an Applied Biosystems model 373A DNA sequencer. Using this approach, we have optimized conditions for the detection of 100 target molecules in a mixture containing 4 micrograms of unrelated DNA. Since the insertion sequence is repeated on average 12-14 times in the genome of M. tuberculosis, this corresponds to a theoretical detection level of 7-8 organisms. Completion of this entire assay can be accomplished in less than 8 h and serves as a basis for further studies in the development of a rapid clinical diagnostic test for tuberculosis.

Base Sequence↗
PubMed1993

Non-radioactive detection of Mycobacterium tuberculosis LCR products in a microtitre plate format.

As part of the development of the ligase chain reaction (LCR) into a tool which can be used by a wide variety of researchers, we have investigated several analytical detection systems for the products of this amplification reaction. While early work with this technology has used gel electrophoresis to separate the LCR probes from the ligated product, solid phase capture techniques are also applicable, particularly when one of the probes is modified with a 'hook' such as biotin, and the adjoining probe modified with a detectable label. In this study we report a comparison of eight different non-radioactive detection techniques and discuss the analytical sensitivity of each. Detection with laser scanning fluorescent gel electrophoresis remains the most sensitive, with the assay described herein capable of detecting 100 molecules of the Mycobacterium tuberculosis insertion element IS6110 in a background of 4 micrograms of unrelated DNA. This method was followed closely by solid-phase capture and chemiluminescence detection which gave a sensitivity of 1000 molecules of IS6110. Fluorescence detection was approximately 10-fold less sensitive than chemiluminescence detection, and absorbance detection was a further 10-fold less sensitive than fluorescence detection. However, absorbance detection even at this level can still be useful for systems where visual interpretation is desired.

Alkaline Phosphatase↗
PubMed1988

Amylase in urine as measured by a single-step chromolytic method.

We studied a new single-step direct chromolytic method (Behring D.A.T.) for measuring urinary amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) activity, comparing results with those by a similar, but two-step, procedure that requires an auxiliary (coupling) enzyme. The two methods gave virtually identical relative responses to purified human pancreatic and salivary amylases. Assay of four quality-control materials to evaluate the total (day-to-day) precision of the new method yielded CVs of 4 to 7%, similar to those of the comparison method for each of the four quality-control samples. Amylase activity was measured by both methods in 110 random (i.e., untimed) urine specimens. Linear regression analysis provided a slope and y-intercept of 0.947 and 4 U/L (x = comparison method, y = direct method), respectively, and a standard error of the estimate of 25 U/L for specimens in which the amylase activities ranged from 11 to 1465 U/L (mean = 358 U/L) by the comparison method. The mean rate of amylase excretion in 2-h timed urine specimens from 95 healthy volunteers, as measured by the new method, was 7.18 (SD = 3.18) U/h, and the nonparametric (95% confidence interval) reference interval was 1.6 to 15.2 U/h. We consider the new method a promising alternative to other kinetic assays that require the use of auxiliary enzymes.

Amylases↗
PubMed1988

Development of a direct assay for alpha-amylase.

We describe a direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. Both human pancreatic and salivary amylase split this substrate without the use of helper enzymes, yielding free 2-chloro-4-nitrophenol, which is monitored at 405 nm. The performance of this reagent compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples. The reagent is very stable in dry powder form and is stable for one month at 2 to 8 degrees C after reconstitution. Because of the rapid color development and linear kinetics (less than 30 s), the assay is easily automated. Results can be obtained in less than 5 min.

Chromatography, High Pressure Liquid↗