An S F, Franklin D, Fleming K A
Nuffield Department of Pathology and Bacteriology, University of Oxford, John Radcliffe Hospital, UK.
Mol Cell Probes. 1992 Jun;6(3):193-200. doi: 10.1016/0890-8508(92)90016-q.
As the polymerase chain reaction (PCR) can be used for the generation of vector-free probes, the optimum conditions for incorporation of digoxigenin-11-dUTP into hepatitis B virus (HBV) probes have been investigated. High yields of double-stranded or single-stranded probes can be obtained by utilizing a pair of primers or one primer alone. The probes were tested by dot-blot hybridization on HBV plasmid DNA, slot-blot hybridization on total cellular RNA of Alexander cells and Southern blot hybridization on cellular DNA of Alexander cells and HBV plasmid DNA. They were also tested by in situ hybridization (ISH) on HBV-positive biopsy liver tissue. A ratio of dig-dUTP:dTTP of 1:3 gave highest sensitivity in DNA hybridization. No loss of amplification efficiency and sensitivity was observed when the final concentration of dig-11-dUTP and dTTP was reduced to 20 microM and 60 microM respectively, compared to 200 microM each of dATP, dCTP, dGTP. Several different sizes of double-strand probes were compared by dot-blot hybridization. Longer probes were more sensitive. Strong signal could also be obtained by combination of two or three small probes, which have overlapping sequences. Single-stranded DNA probes had advantages of simplicity of use, high sensitivity and strand specificity.
由于聚合酶链反应（PCR）可用于生成无载体探针，因此研究了将地高辛素 - 11 - dUTP掺入乙型肝炎病毒（HBV）探针的最佳条件。通过使用一对引物或单独使用一个引物，可以获得高产率的双链或单链探针。通过对HBV质粒DNA进行斑点杂交、对亚历山大细胞的总细胞RNA进行狭缝杂交以及对亚历山大细胞的细胞DNA和HBV质粒DNA进行Southern杂交来检测这些探针。还通过对HBV阳性活检肝组织进行原位杂交（ISH）来检测它们。在DNA杂交中，地高辛 - dUTP与dTTP的比例为1:3时灵敏度最高。与dATP、dCTP、dGTP各为200μM相比，当地高辛 - 11 - dUTP和dTTP的终浓度分别降至20μM和60μM时，未观察到扩增效率和灵敏度的损失。通过斑点杂交比较了几种不同大小的双链探针。较长的探针更灵敏。通过组合两个或三个具有重叠序列的小探针也可以获得强信号。单链DNA探针具有使用简单、灵敏度高和链特异性强的优点。
