ENZYMATIC-SYNTHESIS OF BIOTIN-LABELED POLYNUCLEOTIDES - NOVEL NUCLEIC-ACID AFFINITY PROBES
ENZYMATIC-SYNTHESIS OF BIOTIN-LABELED POLYNUCLEOTIDES - NOVEL NUCLEIC-ACID AFFINITY PROBES
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DOI：
10.1073/pnas.78.11.6633
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发表时间：
1981-01-01
期刊：
影响因子：
--
通讯作者：
WARD, DC
中科院分区：
文献类型：
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作者：
LANGER, PR;WALDROP, AA;WARD, DC
合成了含有生物素分子的dUTP和UTP的类似物，它们通过烯丙胺连接臂共价结合在嘧啶环的C-5位置。这些生物素标记的核苷酸是多种[大肠杆菌、单纯疱疹、小鼠淋巴样肿瘤L1210细胞、人宫颈癌HeLa细胞、禽成髓细胞病病毒逆转录酶]DNA和[小鼠、小牛胸腺、大肠杆菌、T] RNA聚合酶的有效底物。含有低水平生物素取代的多核苷酸（每千碱基50分子或更少）具有变性、再结合和杂交特征，与未取代的对照相似。生物素标记的多核苷酸（单链和双链）被选择性和定量地保留在亲和素- sepharose上，即使经过8 M尿素、6 M胍盐酸盐或99%甲酰胺的大量洗涤。此外，生物素标记的多核苷酸可以在[兔]抗生素素抗体和金黄色葡萄球菌蛋白a存在下选择性免疫沉淀。生物素标记的多核苷酸的独特特征表明，它们将是检测和分离特定DNA和RNA序列的有用亲和探针。
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 were synthesized. These biotin-labeled nucleotides are efficient substrates for a variety of [Escherichia coli, herpes simplex, mouse lymphoid tumor L1210 cells, human cervical carcinoma HeLa cells, avian myeloblastosis virus reverse transcriptase] DNA and [mouse, calf thymus, E. coli, T] RNA polymerases in vitro. Polynucleotides containing low levels of biotin substitution (50 molecules or fewer per kilobase) have denaturation, reassociation, and hybridization characteristics similar to those of unsubstituted controls. Biotin-labeled 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 [rabbit] antibiotin antibody and Staphylococcus aureus protein A. The unique features of biotin-labeled polynucleotides suggest that they will be useful affinity probes for the detection and isolation of specific DNA and RNA sequences.