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Biomedical subjects

Y H Rogers

Publications and source records attributed to Y H Rogers.

6 recordsLinked to original sources
PubMed1999

Immobilization of oligonucleotides onto a glass support via disulfide bonds: A method for preparation of DNA microarrays.

The covalent attachment of disulfide-modified oligonucleotides to a mercaptosilane-modified glass surface is described. This method provides an efficient and specific covalent attachment chemistry for immobilization of DNA probes onto a solid support. Glass slides were derivatized with 3-mercaptopropyl silane for attachment of 5-prime disulfide-modified oligonucleotides via disulfide bonds. An attachment density of approximately 3 x 10(5) oligonucleotides/microm2 was observed. Oligonucleotides attached by this method provided a highly efficient substrate for nucleic acid hybridization and primer extension assays. In addition, we have demonstrated patterning of multiple DNA probes on a glass surface utilizing this attachment chemistry, which allows for array densities of at least 20,000 spots/cm2.

DNA Probes↗
PubMed1997

Nested genetic bit analysis (N-GBA) for mutation detection in the p53 tumor suppressor gene.

There is a growing and significant demand for reliable, simple and sensitive methods for repeated scanning of a given gene or gene fragment for detection and characterization of mutations. Solid-phase sequencing by single base primer extension of nested GBATM primers on miniaturized DNA arrays can be used to effectively scan targeted sequences for missense, insertion and deletion mutations. This paper describes the use of N-GBA arrays designed to scan the sequence of a 33 base region of exon 8 of the p53 gene (codons 272-282) encompassing a hot spot for mutations associated with the development of cancer. Synthetic DNA templates containing various missense, insertion and deletion mutations, as well as DNA prepared from pancreatic and biliary tumor cells, were genotyped using the exon 8 arrays.

Adenocarcinoma↗
PubMed1995

The use of 96-well polystyrene plates for DNA hybridization-based assays: an evaluation of different approaches to oligonucleotide immobilization.

A reliable, simple, and cost-effective method for the immobilization of relatively short (12-30 mer) oligonucleotide probes to 96-well polystyrene plates was required in our laboratory for use in DNA hybridization-based assays. We compared three different approaches to achieve this immobilization. Two of them are modifications of previously published procedures, requiring the use of modified oligonucleotides and/or modified plates. These were compared to a method developed in our laboratory, whereby passive immobilization occurs by incubation in the presence of salt or a cationic detergent. While all methods resulted in the productive binding of the DNA probes and could therefore be used for hybridization, only the passive immobilization approach met our strict performance criteria and was implemented for use in our DNA genotyping laboratory. It was found that the immobilization in the presence of cationic detergents takes place via a unique mechanism.

Base Sequence↗
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

The use of phosphorothioate primers and exonuclease hydrolysis for the preparation of single-stranded PCR products and their detection by solid-phase hybridization.

The effect of phosphorothioate bonds on the hydrolytic activity of the 5'-->3' double-strand-specific T7 gene 6 exonuclease was studied. Double-stranded DNA substrates containing one phosphorothioate residue at the 5' end were found to be hydrolyzed by this enzyme as efficiently as unmodified ones. The enzyme activity was, however, completely inhibited by the presence of four phosphorothioates. On the basis of these results, a method for the conversion of double-stranded PCR products into full-length, single-stranded DNA fragments was developed. In this method, one of the PCR primers contains four phosphorothioates at its 5' end, and the opposite strand primer is unmodified. Following the amplification, the double-stranded product is treated with T7 gene 6 exonuclease. The phosphorothioated strand is protected from the action of this enzyme, whereas the opposite strand is hydrolyzed. When the phosphorothioated PCR primer is 5' biotinylated, the single-stranded PCR product can be easily detected colorimetrically after hybridization to an oligonucleotide probe immobilized on a microtiter plate. We also describe a simple and efficient method for the immobilization of relatively short oligonucleotides to microtiter plates with a hydrophilic surface in the presence of salt.

Base Sequence↗