Harju L, Jänne P, Kallio A, Laukkanen M L, Lautenschlager I, Mattinen S, Ranki A, Ranki M, Soares V R, Söderlund H
Orion Pharmaceutica, Biotechnology, Helsinki, Finland.
Mol Cell Probes. 1990 Jun;4(3):223-35. doi: 10.1016/0890-8508(90)90056-6.
We have devised a sensitive and convenient hybridization technique by combining the polymerase chain reaction (PCR) with affinity-based hybrid collection. In this method 5'-biotinylated primers are used to introduce biotin residues into the DNA fragments during the amplification. The amplified DNA fragments are detected by liquid hybridization using a 32P- or 35S-labelled oligonucleotide as probe. For measurement the hybrids are collected on polystyrene microparticles or onto microtitre wells taking advantage of the biotinavidin interaction. The method is highly sensitive allowing the detection of 30 molecules of DNA. It involves few and simple operations, and is thus suitable for routine diagnostics. The applicability of the method to the detection of HIV-1 DNA from blood, HCMV DNA from urine and HPV-16 DNA from cervical scrapes was evaluated.
我们通过将聚合酶链反应（PCR）与基于亲和的杂交捕获相结合，设计出了一种灵敏且便捷的杂交技术。在该方法中，5'-生物素化引物用于在扩增过程中将生物素残基引入DNA片段。扩增的DNA片段通过使用32P或35S标记的寡核苷酸作为探针的液相杂交进行检测。为了进行测量，利用生物素-抗生物素蛋白相互作用，将杂交体收集在聚苯乙烯微粒或微量滴定板上。该方法高度灵敏，能够检测到30个DNA分子。它涉及的操作很少且简单，因此适用于常规诊断。评估了该方法在检测血液中的HIV-1 DNA、尿液中的HCMV DNA和宫颈刮片中的HPV-16 DNA方面的适用性。
