Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes.

Abstract

Analogs of dUTP and UTP that contain a biotin molecule covalently bound to the C-5 position of the pyrimidine ring through an allylamine linker arm have been synthesized. These biotin-labeled nucleotides are efficient substrates for a variety of DNA and RNA polynerases in vitro. Polynucleotides containing low levels ofbiotin substitution (50'molecules or fewer per kilobase) have denaturation, reassociation, and hybridization characteristics similar. to those of unsubstituted controls. Biotinlabeled polynucleotides, both single and double stranded, are selectively and quantitatively retained on avidin-Sepharose, even after extensive washing with 8 M urea, 6 M guanidine hydrochloride, or 99% formamide. In addition, biotin-labeled polynucleotides can be selectively immunoprecipitated in the presence of antibiotin antibody and Staphylococcus aureus protein A. The unique features ofbiotin-labeled polynucleotides suggest that they will be useful affinity probes for the detection and isolation of specific DNA and RNA sequences.Nucleotide analogs that can function as indicator "probes" when incorporated in polynucleotides would be of significant utility in many procedures used in biomedical and recombinant DNA research. When used in conjunction with immunological, histochemical, or affinity detector systems, such reagents could provide suitable alternatives to radioisotopes for the detection, localization, and isolation of nucleic acid components. Biotin (vitamin H) has many features that make it an ideal probe candidate. The interaction between biotin and avidin, a 68,000-dalton glycoprotein from egg white, has one ofthe highest binding constants (KY., = 10'5) known. (1). When avidin is coupled to appropriate indicator molecules (fluorescent dyes, electrondense proteins, enzymes, or antibodies), minute quantities of biotin can be detected (24). The specificity and tenacity of the biotin-avidin complex has been exploited to develop methods for the visual localization of specific proteins, lipids, and carbohydrates on or within cells (for review, see. ref. 2). Davidson and associates (9-11) chemically crosslinked biotin to RNA, via cytochrome c or polyamine bridges, and used these RNA-biotin complexes as probes for in situ hybridization. The sites of hybridization were visualized in the electron microscope through the binding of avidin-ferritin or avidin-methacrylate spheres. Although this approach to the detection of polynucleotide sequences was successful in the specialized cases examined, a simpler and more general procedure for preparing biotin-substituted nucleic acids was desirable. Biotin directly attached to a nucleotide that functions as an efficient polymerase substrate would be more versatile, both in the experimental protocols and in the detection methods that could be used.We have synthesized a number of nucleotide analogs that contain potential probe determinants (e.g., biotin, iminobiotin, and 2,4-dinitrophenyl groups) covalently attached to the pyrimidine or purine ring in the hope that one of-t...

Analysis of genes and chromosomes by nonisotopic in situ hybridization

“…For applications involving in situ hybridization, enzymatic incorporation of nucleotides modified with biotin [6][7][8][9], digoxigenin [10], dinitrophenol [6,11] or halogenated nucleotides (e.g., BrdU, FrdU) is usually preferred over chemical labeling techniques employing photoreactive Compounds (photobiotin, photodigoxigenin, or photodinitrophenol) because of a higher labeling efficiency. However, other chemical modiftcation schemes using acetylaminofluorene [12], mercuration [13,14], or sulfonation [15] have been used successfully for sensitive nonradioactive detection of hybridized nucleic acid probes [16,17].…”

Flow cytometric detection of β‐globin mRNA in murine haemopoietic tissues using fluorescent in situ hybridization

“…The enzymatic introduction of biotin moieties (19) has had the most widespread application, but also N-Acetoxy-N-Acetylaminofluorene (AAFbmodification (20,26), mercuration (5,151, and sulfonation (30) are being used more and more. In all of the labeling methods mentioned, post hybridization (immunob cytochemical detection is used.…”

Rapid generation of chromosome-specific alphoid DNA probes using the polymerase chain reaction

“…Amplified DNA products were biotinylated with biotin-ll-dUTP by nick translation (Langer et al 1981).…”