Search results for "Restriction enzyme"

Identification of Colletotrichum species responsible for anthracnose of strawberry based on the internal transcribed spacers of the ribosomal region.

2000

In recent years, different molecular techniques have led to an important progress in the characterisation of Colletotrichum species, but there are no available methods which permit the easy identification of Colletotrichum strains and their assignation to classical species. In the present work, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene, were used to identify a total of 80 strains of Colletotrichum, the majority of them isolated from strawberry. One of the most interesting results derived from this study was the easy and reliable distinction, using the endonuclease MvnI, between Colletotrichum fragariae…

Mitochondrial DNA variability of striped dolphins (Stenella coeruleoalba) in the Spanish Mediterranean waters

1995

Frozen muscle samples from 44 striped dolphins stranded on the Spanish Mediterranean coasts from 1990 to 1993 have been studied by means of mitochondrial DNA (mtDNA) restriction site analysis. Thirty-five of these dolphins were affected by a die-off occurring during this time in the western Mediterranean Sea. The mtDNA from each dolphin was digested with 15 restriction endonucleases that recognized 61 different restriction sites. The specific location of these sites on the mitochondrial gene map allowed us to determine the distribution of variability along this molecule. From the restriction analysis, a total of 15 different composite patterns or haplotypes was obtained and their phylogenet…

Assay for O6-alkylguanine-DNA-alkyltransferase using oligonucleotides containing O6-methylguanine in a BamHI recognition site as substrate

1992

Abstract Double-stranded oligonucleotides, 40 bases in length containing an O 6 -methylguanine in a Bam HI restriction site, were developed as substrates for the determination of human O 6 -alkylguanine-DNA-alkyltransferase (AGT). The assay proved highly sensitive and quantitative. After incubation of the 5′-end-labeled oligonucleotides with cell homogenates of peripheral blood lymphocytes, the DNA was digested with Bam HI. Cleavage with this restriction enzyme did not occur in the O 6 -methylguanine-containing oligonucleotide unless the fragment was repaired. The cleaved oligonucleotide was separated from the intact parent oligonucleotide by reverse-phase high-performance liquid chromatogr…

A bicistronic vector backbone for rapid seamless cloning and chimerization of αβT-cell receptor sequences.

2020

To facilitate preclinical testing of T-cell receptors (TCRs) derived from tumor-reactive T-cell clones it is necessary to develop convenient and rapid cloning strategies for the generation of TCR expression constructs. Herein, we describe a pDONR™221 vector backbone allowing to generate Gateway™ compatible entry clones encoding optimized bicistronic αβTCR constructs. It harbors P2A-linked TCR constant regions and head-to-head-oriented recognition sites of the Type IIS restriction enzymes BsmBI and BsaI for seamless cloning of the TCRα and TCRβ V(D)J regions, respectively. Additional well-established TCR optimizations were incorporated to enhance TCR functionality. This included replacing of…

Fine analysis of the chromatin structure of the yeast SUC2 gene and of its changes upon derepression. Comparison between the chromosomal and plasmid-…

1987

Micrococcal nuclease digestion has been used to investigate some fine details of the chromatin structure of the yeast SUC2 gene for invertase. Precisely positioned nucleosomes have been found on a 2 kb sequence from the 3' non-coding region, and four nucleosomes also seem to occupy fixed positions on the 5' flank. Eleven nucleosomes lie on the coding region, although their positioning is not as precise as in the flanks. When the gene is derepressed, these latter nucleosomes adopt a more open conformation and so do two of the nucleosomes positioned on the 5' flank. A dramatic change occurs in the 3' flank, whose involvement in the structural transitions of chromatin upon gene activation is p…

Report of a european collaborative exercise comparing DNA typing results using a single locus VNTR probe

1991

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently…

AMPLIFIED RIBOSOMAL DNA RESTRICTION ANALYSIS (ARDRA) BAKTERI DENGAN POTENSI ANTIMIKROB YANG BERASOSIASI DENGAN SPONS Jaspis sp.

2010

<p><em>Sponges</em><em> are one of the components that compose coral reef which have a potential bioactive substance that has not been utilized. Sponges are generally able to survive in marine waters were nutrients are poor because of associations with other organisms, especially bacteria. This study aimed to isolate and characterize bacteria (endosymbiont and ectosimbion) that produce antimicrobial compounds, and analyze genetic diversity based on Amplified Ribosomal DNA Restriction Analysis (ARDRA). The results of isolation obtained 138 bacterial isolates, which are 70 endofit isolates and 68 surfaces isolates respectively. The results obtained, based on antimicrob…

2014

Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA) included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR) protocol for Insulin-Like Growth Factor 1 (IGF1) detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 3…

Disassembling Papaver: a restriction site analysis of chloroplast DNA

1992

The results from a chloroplast DNA restriction site analysis of the genera Papaver and Roemeria of subf. Papaveroideae (Papaveraceae) and five outgroup taxa are presented. Papaver is represented by 14 species of eight of the 11 sections recognized, Roemeria by two of its three species. Hunnemannia fumariifolia (subf. Eschscholzioideae), Chelidonium majus (subf. Chelidonioideae), Romneya coulteri, Argemone munita and Stylomecon heterophyllum (all subf. Papaveroideae) were chosen as outgroups. DNAs were digested with 24 restriction enzymes. The major results from this analysis are: 1. Papaver, Roemeria and Stylomecon form a monophyletic group supported by at least 17 restriction site mutation…

ABO genotyping by PCR-RFLP and cloning and sequencing

2005

A refined PCR-RFLP based method was established to genotype ABO blood groups. The main objective of this study was to make the techniques also suitable for working with degraded DNA. Specific primer design was carried out to choose fragments shorter than 200 bp as necessary in forensic and archaeological applications. Four fragments of exon 6 and 7 of the ABO gene were amplified and digested by in total 7 restriction endonucleases. Particular attention was paid to the base changes at nucleotide positions 261(delG), 297, 526, 703, 721, 771, 796 and 1060(delC) in order to distinguish the six common alleles A101, A201, B, O01, O02 and O03. Furthermore, this method also enables determination of…