The avidin-biotin complex in bioanalytical applications

Publisher Summary This chapter describes the results of a literature survey showing applications of avidin–biotin technology in tabular forms. One of the tables presents the experimental details from investigations that have used avidin columns for the isolation of target material. Native biotin-containing systems can be isolated directly on such columns. The isolation of other materials, such as membrane proteins and glycol-conjugates, is dependent on the mediation of a biotinylated binder. One of the problems in the use of avidin–biotin technology for isolation purposes is the difficulty encountered in eluting the bound material from the column. Other tables provide information relating to cytochemical localization investigations. These types of investigations usually involve the microscopic visualization of membrane-based sites mediated through the avidin–biotin interaction. In some cases, target sites have been directly biotinylated using group-specific biotinylating reagents. In others, biotinylated lectins, hormones, antibodies, and other binders were employed to mediate between the target molecule and the avidin-probe conjugate or complex.

Applications of avidin-biotin technology: literature survey.

The three different simple repetitive oligonucleotide probes (CT)8, (CAC)5 and (TCC)5 were hybridized to a panel of human DNAs which had been digested with the restriction endonucleases Alu I, Hinf I and Mbo I. The resulting DNA fingerprints were analyzed and different parameters calculated, such as the maximal mean allele frequency and the average number of polymorphic bands per individual. The highest number of bands was obtained after hybridization of Hinf I digested DNA with (CAC)5. The probability of finding the same band pattern as in individual A in individual B is 2 × 10−8. The DNAs of monozygous twins show indistinguishable banding patterns and the bands are inherited according to the Mendelian laws. Thus this procedure reveals informative fingerprints that can be used for individual identification, e. g. in paternity testing and in forensic applications. In most of these experiments 32P-labelled probes were employed, yet the biotinylated oligonucleotide (GACA)4 produced results which were equivalent to those obtained by hybridization with the 32P-labelled probe (GACA)4.

Optimized oligonucleotide probes for DNA fingerprinting

A synthetic 24-mer oligodeoxynucleotide complementary to the region of proopiomelanocortin (POMC) mRNA that codes for the MSH core sequence (αMSH/ACTH[4-11]), ws synthesized and labelled in the 3′-end by use of terminal transferase. Probes tailed with either [3H]- or biotin-labelled nucleotides could be used for in situ hydridization studies. Biotinylated probes, hybridized to mouse and rat pituitary sections, were detected by avidinalkaline phosphatase or streptavidin-alkaline phosphatase procedures and development in 5-brome-4-chloro-3-indolyl phosphate (BCIP)-nitroblue tetrazolium (NBT). Proteinase K pretreatment of sections produced a drastic enhancement of the signal obtained, particularly in strongly fixed, paraffin-embedded material. The non-radioactive in situ hybridization technique compared favourably to radioactive in situ hybridization in terms of rapidity and precision of the localization. Controls involved deletion of the probe to prove that other components of the reaction sequence did not yield stain, digestion with RNase to prove that tissue RNA was necessary to bind the probe, prehybridazation (blocking) with unlabelled probe to prove that the biotinylated probe reacted with its anti-sense region and not its tail and Northern blotting to show that the probe reacted with only one species of pituitary RNA, having the size of mouse pituitary POMC mRNA. In addition, adrenalectomy, known to increase anterior lobe POMC, levels, resulted in both increased numbers and increased intensity of positive corticotroph-like cells. Synthetic oligodeoxynucleotides labelled with biotin appear to constitute attractive reagents for in situ hybridization studies when supported by appropriate control procedures.

Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry.

With a few exceptions DNA probing techniques require the use of radioisotopes and toxic DNA extraction techniques which render the method expensive, potentially hazardous and time-consuming. Most isotopic labeling techniques use the isotope 32P and require 3-10 days to visualize bands after hybridization. An alternative approach is based on the use of non-isotopic detection methods. The available non-isotopic techniques were assessed and their practicality tested. All probes analyzed were tested on samples extracted with a non-toxic extraction procedure using 6 M NaCl as the substitute for phenol and isochloroform. By manipulating probe sizes, blocking agents, selection of membrane and detection system, it is feasible to use non-isotopic labeling and detection in routine parentage testing. Reproducible results were obtained with labeling a variety of DNA probes of various sizes, plasmid and inserts. With an absence of waste disposal costs, probes that are stable for over two years and a staining procedure which takes 3-5 h versus days the technique is well suited for a normal laboratory setting. The next key to the acceptability of DNA testing will be the commercial availability of DNA probes for widespread use.

The use of biotinylated DNA probes in parentage testing: non-isotopic labeling and non-toxic extraction.

Human genomic DNA probes labeled by nick translation with biotin 11-UTP were successfully used to identify single copy restriction fragment length polymorphisms following electrophoresis and Southern blotting onto nylon membranes. Two human probes found on chromosome 21 were used, each of which demonstrated polymorphisms using two separate restriction endonucleases. Compared to isotopic labeling of probes the biotin labels are safer, faster and more economical. The detection method of streptavidin-biotin-alkaline phosphatase permitted phenotyping in a matter of hours after hybridization of the probe to the human DNA fragments.

Joseph Fondell

Papers

The use of biotinylated DNA probes for detecting single copy human restriction fragment length polymorphisms separated by electrophoresis