Chemiluminescence and Bioluminescence
Chapter 9: Gene Assays Based on Bio(Chemi)luminescence
Published:28 Oct 2010
Eleftheria Laios, Penelope C. Ioannou, Theodore K. Christopoulos, 2010. "Gene Assays Based on Bio(Chemi)luminescence", Chemiluminescence and Bioluminescence, Aldo Roda, Pierre Reschiglian
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High detectability, wide analytical range and simple instrumentation constitute the unique advantages of bio(chemi)luminometric methods for DNA/RNA detection and quantification. The development of bio(chemi)luminometric assays performed in microtitration wells allows automation and high sample-throughput, features that are necessary for the routine laboratory. This represents a significant advantage over classical gel electrophoresis, blotting and membrane hybridization. This chapter covers the exploitation of bio(chemi)luminescence in: (a) DNA hybridization assays, (b) quantitative polymerase chain reaction (PCR), (c) genotyping of single nucleotide polymorphisms (SNPs), (d) determination of allele burden, as well as (e) strategies for conjugation of reporter molecules. The methods find a wide range of applications in clinical, environmental and food samples. The assay configurations include: (i) immobilization of the target sequence on a solid surface and hybridization with a probe linked to a reporter, (ii) hybridization of the target to an immobilized probe and subsequent linking of the captured target to a reporter and (iii) hybridization of the target with two probes, one of which is immobilized whereas the other is linked to the reporter. Enzymes (such as alkaline phosphatase, peroxidase, and luciferase), photoproteins (e.g. aequorin), expressible DNA fragments, acridinium esters or nanoparticles can serve as reporters. Immobilization of probes or target sequences is accomplished through the biotin/streptavidin or the hapten/antibody interaction. The bridging of probes or targets with the reporter molecules is carried out either directly by chemical conjugation or indirectly through biotin/streptavidin and hapten/antibody interaction. The development of multianalyte assays is an area of intense research effort.