genomic DNA

About: genomic DNA is a research topic. Over the lifetime, 15046 publications have been published within this topic receiving 663636 citations. The topic is also known as: genomic deoxyribonucleic acid & gDNA.

Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool.

Abstract: In order to develop a technique for distinguishing between isolates of Mycobacterium tuberculosis, we cloned two hypervariable DNA fragments from NdeII-digested genomic DNA. The cloned DNA fragments of 3.8 and 4.7 kb were found to contain the same repetitive element, which was different from previously characterized repetitive elements. It is present in at least 30 copies per genome and is distributed among mycobacterial species other than those of the tuberculosis complex, including M. kansaii, M. gastri, and M. szulgai. When used as a probe on restriction enzyme-digested DNA, it can distinguish between strains from unrelated cases of tuberculosis while demonstrating identical banding patterns for isolates from epidemiologically related cases.

Regional variability in DNA fragmentation after global ischemia evidenced by combined histological and gel electrophoresis observations in the rat brain.

Abstract: We have studied whether the delayed cell death induced by transient forebrain ischemia is associated with an inter-nucleosomal cleavage of DNA into oligonucleosome-sized fragments. The integrity of genomic DNA in various brain regions after a 20-min four-vessel ischemia was examined using gel elec-trophoresis. We found typical ladders of oligonucleosomal DNA fragments in the striatum and in the Ammon's horn. In the latter we also often found a random DNA degradation as a smear pattern. These findings were reinforced by a specific in situ labeling of DNA breaks in tissue sections. A dark staining of nuclei was observed in the cell bodies of neurons—in particular in the head of the caudate and in the vulnerable CAl hippocampal area. With biochemical and histological approaches, there was no evidence of DNA degradation in regions that are resistant to the injury. We conclude that the association of multiple mechanisms of cell damage may occur after a global ischemia. The regional variability in DNA fragmentation stresses the importance of using histological approaches in parallel with gel electrophoresis.

PCR detection of the sheep-associated agent of malignant catarrhal fever.

Abstract: From a genomic library previously constructed from a lymphoblastoid cell line (LCL) propagated from a bovine case of sheep-associated malignant catarrhal fever (SA-MCF), caused by ovine herpesvirus-2 (OHV-2), several OHV-2 clones were identified and characterised by hybridisation using probes from the unique region of the Alcelaphine herpesvirus-1 (AVH-1) genome. Nucleotide sequence from one clone was generated and the predicted amino acid sequence was found to contain regions of homology with the 140 and 160 kDa tegument proteins of Epstein-Barr virus and herpesvirus saimiri respectively. Oligonucleotide primers were constructed and a polymerase chain reaction (PCR) test was developed for the detection of OHV-2 viral DNA. Amplified product was identified by restriction withRsa I andBmy I. The primers were highly specific for OHV-2 DNA with a limit of detection of 6.4 pg of genomic DNA derived from the parent LCL. This was estimated to correspond to one diploid bovine cell. The PCR was successfully applied to detect OHV-2 DNA in peripheral blood leucocytes (pbl) from clinical cases of SA-MCF and normal sheep.

Construction of libraries enriched for sequence repeats and jumping clones, and hybridization selection for region-specific markers

Abstract: We describe a simple and rapid method for constructing small-insert genomic libraries highly enriched for dimeric, trimeric, and tetrameric nucleotide repeat motifs. The approach involves use of DNA inserts recovered by PCR amplification of a small-insert sonicated genomic phage library or by a single-primer PCR amplification of Mbo I-digested and adaptor-ligated genomic DNA. The genomic DNA inserts are heat denatured and hybridized to a biotinylated oligonucleotide. The biotinylated hybrids are retained on a Vectrex-avidin matrix and eluted specifically. The eluate is PCR amplified and cloned. More than 90% of the clones in a library enriched for (CA)n microsatellites with this approach contained clones with inserts containing CA repeats. We have also used this protocol for enrichment of (CAG)n and (AGAT)n sequence repeats and for Not I jumping clones. We have used the enriched libraries with an adaptation of the cDNA selection method to enrich for repeat motifs encoded in yeast artificial chromosomes.