基因,蛋白,免疫 课程6.ppt

Genomics 6 Positional Cloning Key steps in positional cloning Establish pedigrees. The key to the identification of genes responsible for genetic diseases is the identification of pedigrees (large families) in which the disease can be tracked . Obtain biological (blood) samples for provision of DNA for molecular biological analysis from as many members of the pedigree as possible. Link inheritance of disease to inheritance of genetic marker. Refine genetic location using genetic markers for the region of interest. If genetic location is not characterised, use molecular cloning to clone DNA from genetic locus. Derive overlapping DNA sequence from the localised region. Identify genes. Analyse for mutations. Mapping distance by frequency of recombination The aim is to discover how often two loci are separated by meiotic recombination. If two loci are on different chromosomes, they will segregate independently. ie 50% will be recombinant and 50% non -recombinant. This ignores crossovers that occur during meiosis. The closer two markers are located physically, the likelihood that they will be separated by recombination is reduced Recombination fraction defines genetic distance rather than physical distance. Two loci that show 1% recombination are defined as being 1 centimorgan (cM) apart on a genetic map. 1 cM is approximately equal to 1 Mb. Lod score The Lod score is a measure of the likelihood of genetic linkage between loci. For a mendelian character, a lod score greater than +3 is evidence of linkage; one that is less than -2 is evidence against linkage. Types of genetic marker Identification of Candidate Region Linkage analysis in affected families is usually the the first step. Linkage of inheritance with genetic markers throughout the genome Use the mapped location on the chromosome to clone . Essential requirements for mapping genes Sufficient numbers of families to establish linkage. Adequate informative DNA markers. Association of inheritance of disease wi