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Centralized facility on a membrane ressource centre for the European human gene mapping project

Objective


With respect to the European Gene Mapping Project (EUROGEM) the Centre d'Etude du Polymorphisme Humain (CEPH) as Resource Centre for membranes and deoxyribonucleic acid (DNA), had to deliver to all participants the biological material necessary to screen their markers.
For polymerase chain reaction (PCR) analysis, DNA from the 40 CEPH reference families was distributed in microtiterplates (MTP): 5 different families on each MTP. The plates were sent either frozen or with DNA in lyophilized form. For classical hybridizations of Southern membranes, the DNA of the complete panel of the 40 reference CEPH family was digested, checked and then migrated under classical conditions. Most of the operations concerning DNA and reaction buffer distribution were automated. The electrophoretic migration and the electric transfer from the gel to the filter were automatically performed and linked using a prototype of a Multiblotter. Filters were prepared according to requirements with 22 different restriction enzymes. All laboratories received membranes with MspI and Taq1 digests, and a further 3 to 4 membranes with digests of their choice according to the probes they had to use. The quality of all DNA conformed to the reference standards established at CEPH and the filters were optimized. The resource centre for DNA performed and distributed about 100 test filters, 700-parental filters (22 enzymes, 80 parents) and more than 6000 membranes (average 110 per week). The results appeared robust and efficient as data on 576 markers were submitted. These corrected data are now available in CRI-MAP format.
The European Human Gene Mapping Project (EUROGEM) is funded by the European Commission as a collaborative effort to produce a high density linkage map of the human genome. The project is organized as two centralized facilities supplying the 22 network laboratories with the necessary resources (markers and DNA) for linkage studies.

One of these two resource centres is based at the Centre d'Etude du Polymorphisme Humain (CEPH) in Paris. Its role is to supply the participating laboratories with DNA from 40 three-generation families. The other resource centre is based at the Imperial Cancer Research Fund in South Mimms, UK. This centre prepares and supplies the markers to be typed across the families. Two types of markers are supplied: DNA probes for Southern blotting and primers for analysis by polymerase chain reaction (PCR).

In the initial 24 months of the project, over 1800 markers will be screened by the participating laboratories. Approximately half of the markers will be provided by the probe resource centre; the individual laboratories will supply the rest.

In addition to the preparation and distribution of markers, the probe resource centre is also involved in isolating new markers, and in acquiring informative markers, which have been published in scientific literature, from the originators. In the isolation of new markers two approaches are being used. In the first approach, short repeat sequences have been identified from sequence databases and screened for polymorphic length variation by PCR. The second approach is to screen phage and cosmid libraries, either for polymorphic variation of short nucleotide repeats (as above) or with a synthetic decanucleotide probe which detects areas of high variability. The clones identified with this probe are highly likely to detect variable number tandem repeat (VNTR) polymorphisms in Southern blot analysis.

Both screening programmes have been set up and positive clones have been identified for further analysis. The work is expected to lead to the discovery of a number of new markers when the material has been fully analyzed.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

FONDATION JEAN DAUSSET - CENTRE D'ETUDE DU POLYMORPHISME HUMAIN
Address
Rue Juliette Dodu 27
75010 Paris
France