Objective
An important aspect of human genome analysis is the development of detailed physical and genetic maps. The goal is to develop a new methodology which is able to monitor genetic variability on a total genome scale, that is at many variable loci simultaneously, and therefore will facilitate the construction of such detailed maps. Subsequently, this methodology will allow the genetic analysis of inherited phenotypes such as disease, including multifactorial diseases.
Methods are needed to comparatively analyse individual genomes for sequence variation. One way to achieve this is by high resolution 2-dimensional analysis of deoxyribonucleic acid (DNA) fragments, employing both size and sequence separation.
Research was carried out in the following areas:
characterization of a limited set of microsatellite and minisatellite core probes with respect to their suitability for detecting informative variable number tandem repeats (VNTR) loci in the human genome;
development of procedures for direct isolation and cloning of spot giving alleles from 2-dimensional gels;
genetic mapping of a limited set of 2-dimensional spot markers by cosegregation analysis of Centre d'Etude du Polymorphisme Humain (CEPH) pedigrees;
development of procedures for direct hybridization of short sequences to human genomic yeast artificial chromosome (YAC) libraries and cosmid libraries;
development of procedures for in situ mapping of isolated spot giving alleles using primed in situ labelling (PRINS).
For the first time it has been demonstrated that it is feasible to analyse the genome at a large number of loci simultaneously without loss of informativity. Hence, genome scanning for individual differences that could be associated with human disease has now become reality. Industrial applications include the development of automated instrumentation and computer programs to facilitate large scale application and rapid implementation of the system worldwide.
For this purpose saturated genetic and physical maps are necessary in order to detect rapidly genetic linkage in pedigree analysis, and to identify candidate genes in the chromosomal regions defined by the genetic markers. So far, most genetic markers used have been (diallelic) RFLPs, detected by Southern blot analysis as the presence or absence of a particular restriction enzyme recognition site.
More recently, VNTR RFLPs have been described which are detected as variation in length of shortrepeatunit arrays by Southern blot analysis or by PCR. For both these strategies only one locus is analyzed at a time, and for each locus a specific hybridization probe has to be prepared for RFLP analysis or a specific pair of primers synthesized for PCR analysis. The existence of repetitive DNA sequences offers the possibility to assess the mammalian genome in its entirety for individual variation, rather than at one or a few sites. A highly informative set of repeats in this respect are the short tandem repeat sequences (minisatellites and microsatellites) which display VNTR RFLP. With a single oligonucleotide probe (the core probe), between 200 and 700 restriction fragments can be detected, 20 to 40% of which display VNTR polymorphism.
An electrophoretic DNA analysis system, 2-dimensional DNA typing, will be optimized for the simultaneous visualization of all of the DNA restriction fragments detected by a core probe, as spots in a 2dimensional pattern. These patterns will be compared between individuals in the CEPH reference pedigrees to monitor the genetic transmission and segregation of spot variants, perform genetic and physical mapping of the spot variants, and establish their use as marker systems for genetic analysis of human DNA. By comparison with the nearly 2000 genetic markers previously mapped in the reference pedigrees, the 2-dimensional spot variants can be genetically mapped. Physical mapping of spot variants of interest will be done by creating a locusspecific probe by PCR amplification of a fragment from gel or membrane; mapping this locusspecific probe in a specific chromosomal region by in situ labelling on chromosome spreads; and subsequently screening the ICRF YAC gridded reference libraries of the relevant chromosomes in order to determine the contig position of the polymorphic locus.
The creation of a database of 2dimensional markers will, by computerized image analysis, allow the rapid assessment of multiple, highly informative polymorphisms spread over the genome, thereby greatly accelerating the mapping of genetic traits in humans.
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CSC - Cost-sharing contractsCoordinator
2333 CK Leiden
Netherlands