1Lecture 9: Mapping a gene defined by the mutationI. Classical mappingII. Molecular mappingIII. Positional cloning2XA Ba bA Ba bA B a bA B/a ba b/a bA b/a ba B/a bAb + aBtotal= rec, freq.Xa ba b9090101010 + 10200= 0.1 = 10%F1:F2:3Fig. 5.44Fig. 5.555Mapping with a CAPS markerXf EF ef EF ef E F e012116017 X 2= 0.176L C ME/e: CAPS markerF/f: a mutation causing abnormal flowersLer: solid lineCol: dotted lineselfNo. of plantsNo. of recombinant Chr.6A genetic map of DNA markers7Physical mapIn Arabidopsis: 1 cM = about 200 kb (50 genes)In human 1cM = 1000 kb (~17 genes)8Yeast Artificial Chromosomes YACYAC: 100-1000kb yeastBAC: 80-300kb bacteriumCosmid: 20-50 kb bacteriumLamda: 10-20kb bacteriumPlasmid: 0.2-15kb bacteriumVector insert size Host9A MAP OF HUMAN CHROMOSOME 1 Figure 14-7. Linkage map of humanchromosome 1, correlated withchromosome banding pattern. Thehistogram shows the distribution of allmarkers available for chromosome 1.Some markers are genes of knownphenotype, but most are DNA markersbased on neutral sequence variation. Alinkage map, based on recombinantfrequency analyses of the type describedin this chapter, is in the center of theillustration. It shows only some of themarkers available. Map distances areshown in centimorgans (cM, or m.u.).The total length of the chromosome 1map is 356 cM; it is the longest humanchromosome. The positions of somemarkers are cross-referenced to adiagram of subregions of chromosome 1based on a standard banding pattern(such a diagram is called an idiogram).These kinds of correlations can be madeonly by using cytogenetic analysis(Chapter 17) and in situ hybridization.Most of the markers shown on the mapare molecular, but several genes(highlighted in light green) also areincluded.(Griffiths et al (Introduction to geneticanalyses)10Step1: Categorize genes in the regionStep2: Determine mRNA expression patternEST Northern hybridizationStep3: Determine changes between wt and mutExpression patternDNA sequenceStep4: Transgenic studies (SRY example)Case study: Cystic fibrosis CFTR (Cystic fibrosis transmembrane conductance regulator)Steps in identifying the gene in a chromosome region11Fig. 11.19How to determine where the genes are in a segment of DNA?12Fig. 11.20Testes-determining factor (TDF)13Fig. 11.2114Cystic fibrosis is an autosomal recessive disorder thatresults in defective transport of chloride ions throughepithelial cells, and results from mutations in a gene,CFTR, which encodes a cAMP-regulated chloride channel.The primary expression of the defect is in the lungs: asticky mucus secretion accumulates which is prone tochronic infections. Because there are no methods toculture lung cells routinely in the laboratory, in vivo genetherapy approaches have been adopted. As respiratoryepithelial cells are differentiated, retroviral vectors cannotbe used. Instead, gene therapy trials have used adenovirusvectors or liposomes to transfer a suitably sized CFTRminigene, either through a bronchoscope or through thenasal cavity.Cystic fibrosis: a case study15Fig. 11.22aCloning cystic fibrosis transmembrane conductance regulator (CFTR)16Fig. 11.22bNorthern blots help eliminate other 3 candidate genes1717Fig.
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