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Centralized facility for the concerted action on DNA : probe bank for the European human gene mapping project

Objective


The deoxyribonucleic acid (DNA) probe bank facility undertook to distribute approximately 920 markers over the 24 month period. The markers were to be distributed in batches of 5 to 6 markers per laboratory at regular intervals. The actual total weighted value of the markers sent out was 1008 probe equivalents, which slightly exceeded the commitment.

The acquisition of existing DNA probes not tested on Centre d'Etude du Polymorphisme Humain (CEPH) families was initially very important, but it is become less so now, as most new markers published are polymerase chain reaction (PCR) formatted. Also, 36 minisatellite markers were received for inclusion in the project. Their current screening programme has resulted in the detection of a number of polymorphic trinucleotide repeats which will be used at a later stage.
The number of new PCR based markers published are steadily increasing and the informative value is usually very high.

Mononucleotide, dinucleotide, trinucleotide and tetranucleotide repeat sequences have been identified from the European Molecular Biology Library (EMBL) and GenBank sequence databases. The sequences containing repeats were entered into a programme called Primers used to selected PCR primer sets flanking the repeat sequences to that they fulfil certain selection criteria. The PCR primers selected were assessed and those judged most suitable were synthesized. Amplification of human DNA from random unrelated individuals and CEPH families were carried out.
Single chromosome Bluescript libraries were screened for positive clones using a synthetic (AAAT) 15 oligonucleotide.

The task of ensuring that the membranes sent out to each laboratory corresponded to the restriction fragment length polymorphisms (RFLP) detected by the probes sent out has been the responsibility of the Probe Resource Centre.

For data collection 24 Unix workstations, and a Pascal compiler licence were obtained for LINKAGE use and motif was added as an enhanced user interface. The GENBASE software for data management is tightly bound to the project.
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

Imperial Cancer Research Fund (ICRF)
Address
Clare Hall Laboratories Blanche Lane South Mimms
EN6 3LD Potters Bar
United Kingdom