Protocol: DETECTION OF NUCLEIC ACIDS BY HYBRIDISATION

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Tony Rook
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Protocol: DETECTION OF NUCLEIC ACIDS BY HYBRIDISATION

Please find the link for the following protocol:
DETECTION OF NUCLEIC ACIDS BY HYBRIDISATION
By: Ed Rybicki

http://www.mcb.uct.ac.za/manual/hybridn.htm

INTRODUCTION

Hybridisation is a term used to describe the specific complementary association due to hydrogen bonding, under experimental conditions, of single-stranded nucleic acids. It should more properly be referred to as "annealing", as this is the physical process responsible for the association: two complementary sequences will form hydrogen bonds between their complementary bases (G to C, and A to T or U) and form a stable double-stranded, anti-parallel "hybrid" helical molecule. One may make ones nucleic acid single-stranded for the purpose of annealing - if it is not single-stranded already, like most RNA viruses - by heating it in 0.01M NaCl to a point above the "melting temperature" of the double- or partially-double-stranded form, and then flash-cooling to +0oC: this ensures the "denatured" or separated strands do not re-anneal.

Alternatively, one may denature DNA reversibly by treatment with 0.5M NaOH: this does not work for RNA, as this hydrolyses under these conditions.

Summary

A standard hybridisation reaction, then, consists of probing an immobilised target sequence on a membrane with a labelled specific probe sequence: this is done by annealing the probe to the target under (usually) standard "hybridisation conditions" of 0.9M NaCl, 65oC, for 4-16 hr. Probes are usually molecules of DNA or cDNA, a few hundred nt to several kilobases long, cloned into and grown up as recombinant plasmids in E. coli, and purified by caesium chloride gradient centrifugation. One may also use nucleic acid directly purified from the organism of interest, but this is only really effective if this is a virus or a plasmid, as otherwise the probe length is too great, and the repeat number is too small to give appreciable signal. In other words, probes should not be too long, as otherwise one needs very high concentrations of nucleic acid in order to guarantee a sufficient number of copies of the sequence in order to give a detectable "signal" for detection purposes.