Microsatellite markers: isolation with non-radioactive probes and amplification
Arnaud Estoup and Julie Turgeon
The main goal of this technical document (63 pages) is to provide geneticians and ecologists with a reliable method for the isolation of microsatellites and the determination of basic conditions necessary to routinely use them as molecular markers. A constraint related to the isolation of microsatellites is the use of radioactive molecules to screen the partial genomic library. This document offers an alternative to this problem by proposing a screening technique using non-radioactively labelled probes (digoxigenine labelling), an improvement that will most certainly be appreciated by those intending to develop new microsatellite markers. This technical document comprises protocols allowing (1) the quick and reliable cloning of microsatellite markers without the use of any radioactive products, and, (2) the characterisation of microsatellite markers such that conditions for their routine use can be developed. For each task, one or several tests are proposed to allow verifying that each step has been successfully achieved. In order to keep the protocols accessible to those not familiar with molecular techniques, several protocols are also extremely detailed. All of these protocols have been used at the Laboratoire de Genetique des Populations et Evolution of Gif-sur-Yvette (CNRS, FRANCE) and/or at the Laboratoire de Genetique des Poissons of Jouy-en-Josas (INRA, FRANCE). They have been optimised and have proven their efficiency for a number of organisms.
HOW TO DOWNLOAD THIS DOCUMENT:
A zipped file entitled "microev1.zip" (Word 6 format, Windows 3.11) can be downloaded. An original French version of the same document (authors: Arnaud Estoup and Olivier Martin, file "microvf1.zip") is also available. These files are not protected and are available free of charge.
