Day P J, Bevan I S, Gurney S J, Young L S, Walker M R
Department of Clinical Chemistry, University of Birmingham, Edgbaston, U.K.
Biochem J. 1990 Apr 1;267(1):119-23. doi: 10.1042/bj2670119.
The polymerase chain reaction (PCR) has been used to incorporate biotinylated deoxynucleotide triphosphate analogues into a 120 bp sequence from the E6 region of human papilloma virus type 16 (HPV 16). No loss of amplification efficiency is observed utilizing concentrations of up to 200 microM-biotin-11-dUTP, or 180 microM-biotin-7-dATP and -biotin-16-dUTP (where the numbers refer to the number of carbon atoms in the spacer arms). Internally biotinylated PCR products can be detected following slot-blot or vacuum transfer to mitrocellulose or nylon filters without prior electrophoretic separation of the reactants, since unincorporated biotinylated analogues pass through the filter. Internally biotinylated PCR products can also be applied as hybridization probes in Southern blot analysis or in situ hybridization. This system enables detection of PCR products or target sequences at levels below that for 5'-biotinylated probes and can be applied in an 'open sandwich assay' without the need for a separate labelled probe currently required in conventional sandwich assays. However, as hybridization probes, sensitivity may be limited by the steric hindrance of strand hybridization possibly due to the spacer arms linking the nucleotides to the biotin molecule.
聚合酶链反应（PCR）已被用于将生物素化的脱氧核苷三磷酸类似物掺入人乳头瘤病毒16型（HPV 16）E6区域的一段120 bp序列中。使用浓度高达200 μM的生物素-11-dUTP、180 μM的生物素-7-dATP或生物素-16-dUTP（数字表示间隔臂中的碳原子数）时，未观察到扩增效率的损失。由于未掺入的生物素化类似物会穿过滤膜，因此在将反应物进行槽式印迹或真空转移至硝酸纤维素或尼龙滤膜后，无需事先对反应物进行电泳分离，即可检测内部生物素化的PCR产物。内部生物素化的PCR产物还可作为杂交探针用于Southern印迹分析或原位杂交。该系统能够检测低于5'-生物素化探针水平的PCR产物或靶序列，并且可用于“开放夹心测定”，而无需传统夹心测定中目前所需的单独标记探针。然而，作为杂交探针，灵敏度可能会受到链杂交空间位阻的限制，这可能是由于连接核苷酸与生物素分子的间隔臂所致。
