Wahlberg J, Lundeberg J, Hultman T, Holmberg M, Uhlén M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm.
Mol Cell Probes. 1990 Aug;4(4):285-97. doi: 10.1016/0890-8508(90)90020-z.
We describe a rapid solid phase assay for detection and sequencing of DNA sequences based on selective introduction of biotin and isotope into the specific DNA fragment amplified by the polymerase chain reaction (PCR). A two-step PCR procedure is used to lower the background signal. The in vitro amplified material is immobilized on magnetic beads with covalently coupled streptavidin and the amount of bound label is measured. Samples identified as positive can be analysed by direct solid phase DNA sequencing. A strategy is also described to use general primers for detection, capturing and sequencing, which are not homologous to the specific sequence to be detected. The concept has been optimized using oligonucleotides specific for Staphylococci and Streptococci, respectively. Here, we show that the assay can be used for detection of Plasmodium falciparum in clinical samples.
我们描述了一种基于在聚合酶链反应（PCR）扩增的特定DNA片段中选择性引入生物素和同位素来检测和测序DNA序列的快速固相测定法。采用两步PCR程序以降低背景信号。体外扩增的材料固定在共价偶联抗生物素蛋白的磁珠上，并测量结合标记物的量。鉴定为阳性的样品可通过直接固相DNA测序进行分析。还描述了一种使用通用引物进行检测、捕获和测序的策略，这些引物与要检测的特定序列不同源。该概念已分别使用针对葡萄球菌和链球菌的寡核苷酸进行了优化。在此，我们表明该测定法可用于检测临床样品中的恶性疟原虫。
