Fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) is a rapid and reliable technique for chromosomal investigations that is used for a wide variety of cytogenetic purposes at present. This molecular-cytogenetic method has been developed continuously for many years. As a consequence, various modifications with different kinds of fluorescently labelled probes have been introduced to optimise the detection of DNA and RNA sequences. This review articlepaper presents the general principles of in situ hybridisation, probe labelling and examples of proper use of different kinds of probes. In addition, some newer FISH methods and their usefulness in human molecular cytogenetics are described.

QUANTITATIVE DETECTION OF TRANSGENIC AND ENDOGENOUS DNA SEQUENCES IN SEEDS AFTER AUTOMATED DNA PREPARATION

In applied transgenic research as well as in agriculture there is an increasing need for high-throughput analyses of plants for genotypic selection or to identify the purity of seed stocks, e.g. for transgenic contaminations or the identification of pathogens. We developed and optimised conditions for the isolation of DNA from single seeds using an automated high-throughput protocol. Our results show that the system provided is capable of isolating DNA from any tested seed source. Furthermore, seeds remain capable of germinating during the homogenisation procedure. Quantification of endogenous and transgenic sequences by Real-Time PCR revealed that the prepared DNA is suitable in quality and quantity for multiple PCR analyses with both SYBR Green and hybridisation probe detection. The described method will be a useful tool for routine analyses like screening of large populations or the specific detection of genetically modified organisms (GMO).