Nucleic acid analysis by sandwich hybridization.
One of the most significant achievements of the biochemist during the past two decades is the use to which immunologically based assays have been put in clinical diagnosis (Hood et al.: Immunology, 1984). The problem faced and surmounted by immunologists in effecting the transition from research tool to routine clinical assay bears a remarkable similarity to that confronting the molecular biologist today; i.e., how can nucleic acid hybridization, a technique of obvious potential (Meinkoth and Wahl: Anal Biochem 138:267-284, 1984; Syvanen: Med Biol 64:313-324, 1986; Matthews and Kricka: Anal Biochem 169:1-25, 1988), be modified in order to fulfill all necessary parameters of a routine diagnostic assay? There are several such requirements, and the importance placed on each depends on the objectives of the assay: the technique must be sensitive, specific, and reproducible. Other advantages would be cost-effectiveness, ease of manipulation, and amenability to automation. Ideally, the signal detection should be based on a non-radioactive system, because of the instability of probes labelled with isotopes like 32p, and the potential hazards involved in their handling and disposal. The sandwich hybridization for the analysis of nucleic acid sequences was first used in 1977 (Dunn and Hassell: Cell 12:23-36, 1977), but its potential as a diagnostic assay was not realized until 1983, when it was applied to the detection of adenovirus DNA in nasopharyngeal aspirates from children with acute respiratory infection (Ranki et al: Gene 21:77-85, 1983). It has since been modified and used not only for the detection of microbial infection (Virtanen et al.: Lancet i:381-383, 1983; Ranki et al.: Cur Top Microbiol Immunol 104:307-318, 1983; Lehtomaki et al.: J Clin Microbiol 24:108-111, 1986; Virtanen et al.: J Clin Microbiol 20:1083-1088, 1984; Palva and Ranki: Clin Lab Med 5:475-490, 1985; Polsky-Cynkin et al.: Clin Chem 31:1438-1443, 1985; Parkkinen et al.: J Med Virol 20:279-288, 1986; Palva: FEMS Microbiol Lett 28:85-91, 1985; Palva et al: FEMS Microbiol Lett 23:83-89, 1984; Zolg et al.: Mol Biochem Parasitol 22:145-151, 1987; Palva: J Clin Microbiol 18:92-100, 1983), but also for the analysis of nucleotide sequence variations (Langdale and Malcolm: Gene 36:201-210, 1985). We will discuss the development of the sandwich technique and the advantages it conveys over the more conventional nucleic acid hybridization formats, together with new developments which will ensure that it earns a place alongside immunoassay in the diagnostic laboratory.
Nicholls
P J
PJ
Department of Biochemistry, Charing Cross and Westminster Medical School, London, England.
Malcolm
A D
AD
D000818:Animals; D001424:Bacterial Infections / Q000175:diagnosis / Q000235:genetics; D002690:Chlamydia Infections / Q000175:diagnosis / Q000235:genetics; D002692:Chlamydia trachomatis / Q000235:genetics; D004247:DNA / Q000032:analysis*; D006801:Humans; D007239:Infections / Q000175:diagnosis* / Q000235:genetics; D008288:Malaria / Q000175:diagnosis / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010963:Plasmodium falciparum / Q000235:genetics; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D012313:RNA / Q000032:analysis*; D014777:Virus Diseases / Q000175:diagnosis / Q000235:genetics
D012313:RNA; D004247:DNA