3-5.8Detection of non-radioactively labeled probes after hybridization:
Affinity molecules (streptavidin or digoxigenin-specific antibody) are conjugated with a variety of marker groups or molecules. They include various fluorophores or enzymes such as alkaline phosphatase and peroxidase which can permit detection via colorimetric assays, chemical luminescence assays or fluorescent assay.
In colorimetric assays, alkaline phosphatase catalyzes removal of phosphate group from BCIP (5-bromo-4-chloro-3-indolyl phosphate), generating a product that dimerizes to di-bromo-di-chloro indigo, which reduces NBT (Nitrobluetetrazolium) to insoluble purple dye, diformazan that becomes visible at sites where probe has hybridized.
Fluorescent assays make use of HNPP (2-hydroxy-3-naphthoic acid 2'-phenylanilide phosphate). After de-phosphorylation by alkaline phosphatase, HNPP generates fluorescent precipitate on membranes that can be excited by irradiation at 290nm. The response signal emitted at 509nm are captured by CCD cameras.
Chemiluminescence is the fastest and most sensitive assay using HRP (Horseradish peroxidase) – luminal detection system. HRP catalyzes the oxidation of luminal in the presence of H2 O2 , generating reactive peroxide that emits light at 425nm during decomposition to its ground state.
Applications: This technique is used in physical mapping, karyotyping and phylogenetic analysis, gene expression profiling in developmental biology, pathogen profiling, and abnormal gene expression in pathology and for morphology and population structure of microorganisms.
BOOK
Strachan T and Andrew P Read. Human Molecular Genetics,2nd edition.
Samrook J and David W. Russell Molecular Cloning:A Laboratory Manual, 3rd Edition.
BIBLIOGRAPHY
Eleftherios P. Diamand, Theodore K. Christopoulos (1991). The Biotin-(Strept)Avidin System: Principles and Applications in Biotechnology. CLIN. CHEM. 37/5, 625-636.
Grunstein M., David S.,Hogness (1975). Colony hybridization: A method for the isolation of clofied DNAs that contain a specific gene. Proc. Nat. Acad. Sci. USA Vol. 72, No. 10, pp. 3961-3965.
BlouinJ. L., Rahmani Z., ChettouhZ., PrieurtM., FermaniantJ et al1990. Slot Blot Method for the Quantification of DNA Sequences and Mapping of Chromosome Rearrangements: Application to Chromosome 21. Am. J. Hum. Genet. 46:518-526.
Pennina R. Langer-Safer, Michael Levine, David C. Ward(1982). Immunological method for mapping genes on Drosophila polytene chromosomes. Proc. NatL Acad. Sci. USA Vol. 79, pp. 4381-4385.
Marilena Aquino de Muro. Probe Design, Production, and Applications. Medical Biomethods Handbook 2005, pp 13-23.