ISOLATION AND LOCALIZATION OF 3 NEW RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) MARKERS TO UNIQUE POSITIONS ON THE SHORT ARM OF HUMAN CHROMOSOME 3

Abstract

The construction of a genetic linkage map depends upon the detection of recombination events that occur between homologous chromosomes during meiosis. These recombination events may be directly observed in spermatozoa (Morton, et. al., 1982) or indirectly observed by following the inheritance pattern of DNA markers in three generation families. The latter method of mapping became feasible in humans with the discovery of Restriction Fragment Length Polymorphisms (RFLPs). The discovery of these randomly spaced markers has resulted in two linkage maps of the human genome (Donis-Keller, et. al., 1987; Weissenbach, et. al., 1992), which will serve as the foundation for the eventual construction of the physical map and the determination of the nucleotide sequence of the human genome (National Research Council, 1988). The short arm of human chromosome 3 has been the object of intensive research with the discovery that this chromosomal arm harbors numerous genes believed to play a role in human disease. The construction of a high density genetic linkage map of human chromosome 3p (Tory, et. al., 1992) is a crucial step in the isolation and cloning of these putative disease genes. The three single copy fragments localized in this analysis were first isolated from two independent chromosome 3 flow sorted phage libraries (LAO3NS01 and LAO3NS02) which had been screened to eliminate most repetitive human sequences. The human chromosome 3 specific inserts were isolated from the recombinant Charon 21A vector by Eco RI digestion, gel purified, radiolabeled using the random priming method (Feinberg and Vogelstein, 1983) and analyzed against two somatic cell hybrid screening panels by Southern hybridizations. The first screen distinguished hamster from human sequences and human 3p from 3q sequences. The second screen localized the human 3p sequences to five discreet regions of the chromosome. These sequences were selected for further analysis. The selected sequences were tested for their ability to detect frequent RFLPs by Southern hybridizations against genomic digests of five unrelated individuals whose DNA had been digested with ten different endonucleases (Aldridge, et. al., 1984). Those sequences that detected frequent RFLPs were next hybridized against genomic digests of twenty individuals to determine their observed Frequency of Heterozygosity (FOH). Those sequences with an observed FOH of .30 or greater were selected for final localization.