Wahlberg J, Lundeberg J, Hultman T, Uhlén M
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
Proc Natl Acad Sci U S A. 1990 Sep;87(17):6569-73. doi: 10.1073/pnas.87.17.6569.
A system for rapid colorimetric detection of specific genome DNA fragments amplified by the polymerase chain reaction (PCR) is described that has been designed to allow direct solid-phase sequencing of positive samples. The amplified material is immobilized on magnetic beads by using the biotin streptavidin system. An Escherichia coli lac operator DNA sequence is incorporated in the amplified material during the second step of a nested primer procedure. This 21-base-pair sequence is used for a general colorimetric detection with a fusion protein consisting of the E. coli Lac repressor and beta-galactosidase. Positive samples can be treated subsequently with alkali to obtain a single-stranded DNA template suitable for direct genomic sequencing. This method to detect immobilized amplified nucleic acids (DIANA) is well adapted for automated or semiautomated clinical assays. Here, we show that it can be used to detect and sequence Chlamydia trachomatis genomic DNA in clinical samples.
本文描述了一种用于快速比色检测经聚合酶链反应（PCR）扩增的特定基因组DNA片段的系统，该系统旨在允许对阳性样本进行直接固相测序。通过生物素-链霉亲和素系统将扩增产物固定在磁珠上。在巢式引物程序的第二步中，将大肠杆菌乳糖操纵子DNA序列掺入扩增产物中。这个21个碱基对的序列用于与由大肠杆菌乳糖阻遏物和β-半乳糖苷酶组成的融合蛋白进行通用比色检测。阳性样本随后可用碱处理以获得适合直接基因组测序的单链DNA模板。这种检测固定化扩增核酸的方法（DIANA）非常适合自动化或半自动化临床检测。在此，我们表明它可用于检测临床样本中的沙眼衣原体基因组DNA并进行测序。
