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Biomedical subjects

I Durrant

Publications and source records attributed to I Durrant.

At least 19 recordsLinked to original sources
PubMed1997

Labeling of oligonucleotides with fluorescein.

The labeling of oligonucleotide probes using a fluorescein-labeled nucleotide is described. The reaction is characterized by careful control of the nucleotide and probe molar ratio in order to produce a tail that gives good detection sensitivity without compromising hybridization stringency control of the probe sequence. The labeling reaction can be easily monitored for incorporation of the fluorescent label and the probes can be used in many applications.

DNA↗
PubMed1996

Preparation of horseradish peroxidase-labeled probes.

The direct labeling of nucleic acid probes with horseradish peroxidase (HRP) may be used in many membrane hybridization applications, including Southern blots, Northern blots, colony and plaque screening, PCR products detection/identification. This article describes the preparation method, which involves the labeling of a single-stranded nucleic acid probe with a positively charged HRP-parabenzoquinone-polyethyleneimine complex (labeling reagent). The associated hybridization and posthybridization protocols are relatively simple, which makes probes labeled directly with HRP particularly suitable for large scale screening, where tens or hundreds of blots are processed weekly.

Blotting, Northern↗
PubMed1996

Hybridization of horseradish peroxidase-labeled probes and detection by enhanced chemiluminescence.

This article describes the use of probes directly labeled with horseradish peroxidase in conjunction with enhanced chemiluminescence, which allows a flexible approach to hybridizations and detections. This system may be used with the following applications: Southern blots, Northern blots, colony and plaque screening for positive clones, YAC clone screening, and PCR products detection. The major steps required for the use of directly labeled HRP probes are hybridization, stringent washes, and detection.

DNA Probes↗
PubMed1996

Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

Biopsy↗
PubMed1995

Evaluation of novel formulations of 35S- and 33P-labelled nucleotides for in situ hybridization.

Radioactive in situ hybridization predominantly utilizes either RNA probes or oligonucleotide probes. The properties of various formulations of [35S]UTP alpha S have been studied with respect to probe labelling and when applied to in situ hybridization. A new formulation has been prepared that combines a high physical concentration with a high specific activity so that, theoretically, predominantly full-length, high-specific-activity primary transcripts are produced. [alpha-33P]UTP can also be used for in situ hybridization and it compares favourably to 35S-labelled probes in terms of resolution and sensitivity. Both radiolabels can also be used to label oligonucleotide probes by a tailing reaction. [35S]dATP alpha S has been reformulated specifically for labelling oligonucleotides. The traditional stabilizer, dithiothreitol, which may cause precipitation within the tailing reaction buffer, has been replaced with an alternative stabilizer that avoids this problem while maintaining the stability of the nucleotide.

Adenosine Triphosphate↗
PubMed1995

Fluorescein as a label for non-radioactive in situ hybridization.

Non-radioactive techniques can be applied to many in situ hybridization (ISH) applications, and a number of non-radioactive labels for this process have been reported. However, these labels have some inherent problems in terms of both background and signal-to-noise values. We have sought to address these issues by searching for an alternative label that has the following features: efficient incorporation into probes, non-endogenous to biological systems, the availability of a high-affinity, high-specificity antibody. Fluorescein has been shown to meet these requirements. In addition, due to the fluorescent nature of the label, it has been possible to design a rapid, non-radioactive labelling assay and also to view in situ hybridization results by direct fluorescence in certain ISH applications. The hybridization kinetics have been investigated. Significant improvements have been made to the hybridization buffer leading to reduced background and increased rates of hybridization when compared to traditional hybridization buffers.

Animals↗
PubMed1991

Enhanced chemiluminescence for the detection of membrane-bound nucleic acid sequences: advantages of the Amersham system.

A range of nonradioactive nucleic acid labeling and detection systems have been developed that enable the user to label probes directly with enzyme molecules or indirectly with hapten-derivatized nucleotides. Horseradish peroxidase is used for the direct labeling procedures due to the ease of chemical modification and the relative thermal and chemical stability of this enzyme. Horseradish peroxidase has also been conjugated to a high-specificity antifluorescein antibody for detection of hapten (fluorescein)-labeled hybrids. Enhanced chemiluminescence is a light-emitting process optimized for the detection of low levels of horseradish peroxidase on membrane supports. Results are obtained as hard copy images on x-ray film.

Base Composition↗