The biotin-(strept)avidin system has been used for many years in a variety of different applications. Here we present a general overview of the system, describe its components and advantages, and show how the system is used in various applications, with emphasis on immunological and nucleic acid hybridization assays. This system is now considered a versatile independent technology with broad applications in many branches of biotechnology. Clearly, its use will continue to grow in the years to come.

The biotin-(strept)avidin system: principles and applications in biotechnology.

The characterization of genetic variation at the DNA level has generated significant advances in gene and disease mapping1, and in the forensic identification of individuals2–6. The most common method of DNA analysis, that of restriction fragment length polymorphism (RFLP), requires microgram amounts of relatively undegraded DNA for multi-locus typing, and hundreds of nanograms for single-locus comparisons7. Such DNA frequently cannot be obtained from forensic samples such as single hairs and blood stains, or from anthropological, genetic or zoological samples collected in the field. To detect polymorphic DNA sequences from single human hairs, we have used the polymerase chain reaction (PCR), in which specific short regions of a gene can be greatly amplified in vitro8–10 from as little as a single molecule of DNA10. We have detected genetically variable mitochondrial and nuclear DNA sequences from the root region of shed, as well as freshly-plucked, single hairs; mitochondrial DNA (mtDNA) sequences have been detected in a sample from a single hair shaft. We have used three different means of DNA typing on these samples: the determination of amplified DNA fragment length differences, hybridization with allele-specific oligonucleotide probes, and direct DNA sequencing.

DNA typing from single hairs

Isolation, characterization, and expression in Escherichia coli of the DNA polymerase gene from Thermus aquaticus.

Oligonucleotides bind specifically to single-stranded nucleic acids to form a double helix if there is a complementary antiparallel nucleotide sequence. In addition, certain oligonucleotides bind specifically to a variety of proteins. Therefore, biological processes involving these nucleic acids or proteins can be modulated (normally inhibited) by addition of the respective oligonucleotides. The effects of these oligonucleotides and the fields of potential application can be broadened by the introduction of chemically modified nucleotides. For instance, replacing oligonucleotide phosphate groups by methylphosphonates results in the loss of one negative charge per nucleotide and the oligonucleotide becomes more lipophilic. An oligonucleotide carrying a reactive group can modify its binding partner. An oligonucleotide covalently linked to a dye can be localized in a biological specimen. Oligonucleotides attached to an enzyme can be detected in very small amounts since the enzyme can catalyze the formation of large amounts of the substance assayed (e.g., a fluorescent product). An important biochemical application is the detection and localization of a messenger RNA or its gene. Medical applications include the detection of bacterial or viral sequences. There is also great interest in inhibiting the translation of messenger RNA and the transcription and replication of DNA. So-called antisense oligonucleotides are currently being used for the inhibition of protein biosynthesis and of reverse transcription of retroviruses.

Chemically Modified Oligonucleotides as Probes and Inhibitors

Molecular biology has revolutionized the understanding of many aspects of human disease. Ongoing developments in DNA diagnostics--the analysis of disease at the nucleic acid level--will soon provide automated, rapid, and inexpensive analyses for DNA or RNA sequences associated with genetic, malignant, and infectious diseases. DNA diagnostics will also facilitate the identification of disease-associated genes at birth, thus creating new opportunities for preventive medicine.

https://science.sciencemag.org/content/sci/242/4876/229.full.pdf

DNA Diagnostics--Molecular Techniques and Automation

Previously, DNA polymorphisms in the HLA gene cluster have been analyzed using radioactive probes in Southern blot experiments; the restriction fragment length polymorphisms (RFLPs) revealed by this analysis are capable of subdividing HLA serological types. Here, we report the use of DNA probes labeled with biotinylated psoralen to provide nonisotopic detection of HLA class II RFLP patterns. These biotinylated probes contain cDNA sequences encoding the alpha and beta chains of DP, DQ, and DR HLA class II genes as inserts in M13 vectors. The recombinant M13 molecules are partially double-stranded with single-stranded HLA cDNA regions and contain biotinylated psoralen covalently linked to duplex DNA by UV irradiation. Following hybridization, the presence of biotinylated probe bound to target DNA is detected using a streptavidin-horseradish peroxidase conjugate, which converts the colorless substrate 3,3',5,5'-tetramethylbenzidine to a blue precipitate in less than 1 hr. The probe and detection system described here can detect single-copy genes in less than 0.5 microgram of total human DNA on Southern blots and generates the same specific RFLP patterns as do probes labeled with 32P by nick-translation. These biotinylated HLA class II probes have been applied to tissue typing for bone marrow transplantation and the study of insulin-dependent diabetes susceptibility, revealing in each case relevant polymorphisms not detected by serologic typing.

Use of nonisotopic M13 probes for genetic analysis: application to HLA class II loci.

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Use of nonisotopic M13 probes for genetic analysis: application to HLA class II loci.