Search PubMed⌕ Search

Biomedical subjects

G L Trainor

Publications and source records attributed to G L Trainor.

8 recordsLinked to original sources
PubMed1994

Genetic Bit Analysis: a solid phase method for typing single nucleotide polymorphisms.

A new method for typing single nucleotide polymorphisms in DNA is described. In this method, specific fragments of genomic DNA containing the polymorphic site(s) are first amplified by the polymerase chain reaction (PCR) using one regular and one phosphorothioate-modified primer. The double-stranded PCR product is rendered single-stranded by treatment with the enzyme T7 gene 6 exonuclease, and captured onto individual wells of a 96 well polystyrene plate by hybridization to an immobilized oligonucleotide primer. This primer is designed to hybridize to the single-stranded target DNA immediately adjacent from the polymorphic site of interest. Using the Klenow fragment of E. coli DNA polymerase I or the modified T7 DNA polymerase (Sequenase), the 3' end of the capture oligonucleotide is extended by one base using a mixture of one biotin-labeled, one fluorescein-labeled, and two unlabeled dideoxynucleoside triphosphates. Antibody conjugates of alkaline phosphatase and horseradish peroxidase are then used to determine the nature of the extended base in an ELISA format. This paper describes biochemical features of this method in detail. A semi-automated version of the method, which we call Genetic Bit Analysis (GBA), is being used on a large scale for the parentage verification of thoroughbred horses using a predetermined set of 26 diallelic polymorphisms in the equine genome.

Autoanalysis↗
PubMed1994

Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate.

Biodistribution and metabolism of oligonucleotides were determined using a 3H-labeled 20-nucleotide phosphodiester and its phosphorothioate analog. The oligonucleotides were radiolabeled by 3H-methylation of an internal deoxyctidine with HhaI methylase and S- [3H]adenosylmethionine. Biodistribution studies were conducted after intravenous injection of 6 mg/kg (5 muCi) oligonucleotide. Metabolism of the oligonucleotides was determined by paired-ion high performance liquid chromatography. After phosphodiester injections, radiolabel rapidly cleared the blood. Relative initial concentrations were as follows: kidney > blood > heart > liver > lung > spleen. Radiolabel in spleen peaked at 1 hr and remained elevated for 24 hr. At 2 hr the concentration in all organs, except spleen, was equal to that in blood. High performance liquid chromatographic analysis of the kidney, liver, and spleen extracts and urine indicated extremely rapid metabolism to monomer. Results of studies after the injection of phosphorothioate oligonucleotide differed from those using the phosphodiester. Despite its rapid clearance from blood, phosphorothioate accumulated rapidly in all tissues, especially the kidney. Kidney uptake increased over time, remaining very high for 24 hr. Ratios of organ to blood concentrations at 2 hr for all organs were 5:1 or greater. Kidney and liver ratios were 84:1 and 20:1, respectively. Analysis of the kidney and liver extracts and urine indicated that slow metabolism occurred. These data suggest that phosphodiester oligonucleotides would have limited therapeutic utility. The stability and organ distribution of the phosphorothioate oligonucleotide imply that such oligonucleotides may have therapeutic potential.

Animals↗
PubMed1992

DNA recognition of base analogue and chemically modified substrates by the TaqI restriction endonuclease.

It has been proposed that protein-DNA recognition is mediated via specific hydrogen bond, hydrophobic, and/or electrostatic interactions between the protein and DNA surfaces. We have attempted to map and quantitate the energies of these interactions for the TaqI endonuclease by constructing substrates substituted with base or phosphate analogues that either remove or sterically obstruct particular functional groups in the canonical TCGA sequence. The DNA backbone was also modified using a chemical approach (phosphate ethylation) which identified several phosphates in the recognition sequence essential for cleavage. The base analogues, N6-methyl-A, N7-deaza-A, N7-deaza-G, inosine, N4-methyl-C, 5-methyl-C, uracil, 5-bromo-U, and the phosphate analogues, alpha-thio-A, alpha-thio-G, alpha-thio-T, alpha-thio-A, were substituted for their corresponding unmodified counterpart in one strand of the TCGA duplex. The effects of these analogues were monitored by measuring the steady state (Km, kcat) and single-turnover (kst) kinetic constants. Only the N6-methyl-A-substituted DNA, which mimics in vivo methylation, was unreactive while the remaining analogue substitutions exhibited Michaelis-Menten kinetics. In general, the Km was either unchanged or lowered by the analogue substitutions. In contrast, many of the analogues severely reduced kcat, suggesting the modified functional groups served mainly to destabilize the transition state. Single-turnover measurements paralleled the kcat results, pointing to the N7 and N6 of A, the N7 of G, and one of the nonbridging oxygens 3' to T as putative contacts made in achieving the transition state. Substrates with double substitutions displayed simple additivity of delta delta G" implying that these changes behaved independently. The unmodified strand in 10 out of 12 hemisubstituted substrates had a normal kst value suggesting that a particular cleavage center is controlled predominantly by recognition of determinants on the same strand as the scissile bond. These results are discussed in relation to base analogue work from the EcoRI, RsrI, and EcoRV restriction endonucleases.

Base Sequence↗
PubMed1991

Improvements in the chain-termination method of DNA sequencing through the use of 7-deaza-2'-deoxyadenosine.

Significant improvements in the quality of DNA sequencing data have been shown when deoxyadenosine triphosphate (dATP) is replaced by 7-deaza-2'-deoxyadenosine triphosphate (c7dATP). The use of c7dATP in conjunction with 7-deaza-2'-deoxyguanosine triphosphate (c7dGTP) further decreases anomalies in electrophoretic mobility which are caused by compressions involving G and/or A residues. This effect is observed for both isotope-based and fluorescence-based sequencing approaches. Replacing dATP with c7dATP also results in a higher degree of uniformity in the frequency of chain termination reactions, when such terminations involve the incorporation of fluorescence-labeled dideoxynucleotides by T7 polymerase. These improvements in the gel-resolution and distribution of chain-terminated DNA products result in higher accuracy in both manual and automated base assignment.

Adenosine Triphosphate↗
PubMed1990

DNA sequencing, automation, and the human genome.

DNA sequencing is one of the key analytical operations of modern molecular biology and a crucial element of biotechnology. The principles of DNA sequencing and details of the technologies of both manual, radioisotope-based and automated, fluorescence-based approaches are described. The goals and rationale of the Human Genome Initiative are discussed along with implications for future sequencing technologies. Finally, a glimpse of emerging DNA sequencing technologies is offered.

Autoanalysis↗
PubMed1987

A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides.

A DNA sequencing system based on the use of a novel set of four chain-terminating dideoxynucleotides, each carrying a different chemically tuned succinylfluorescein dye distinguished by its fluorescent emission is described. Avian myeloblastosis virus reverse transcriptase is used in a modified dideoxy DNA sequencing protocol to produce a complete set of fluorescence-tagged fragments in one reaction mixture. These DNA fragments are resolved by polyacrylamide gel electrophoresis in one sequencing lane and are identified by a fluorescence detection system specifically matched to the emission characteristics of this dye set. A scanning system allows multiple samples to be run simultaneously and computer-based automatic base sequence identifications to be made. The sequence analysis of M13 phage DNA made with this system is described.

Automation↗