Lecture 20 Chapter 20

Lecture 20 Chapter 20 - PowerPoint Slides for Introduction...

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1 PowerPoint Slides for Introduction to Genetic Analysis Ninth Edition Anthony J. F. Griffiths, Susan R. Wessler, Richard C.  Lewontin, and Sean B. Carroll CHAPTER 20 Gene Isolation and Manipulation Copyright 2008 © W H Freeman and Company
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2 Cloning Disease Genes 1. Positional Cloning 1. Cloning based on cross-species  homolog
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3 Positional Cloning 1. Map disease to a specific chromosomal region.  1. Through recombination analysis and linkage disequilibrium. 1. Fine tune localization  1. Linkage analysis using a high resolution map of known markers (DNA  polymorphisms in the region). 1. Capture the region  1. Through chromosome walking.
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4 Positional Cloning of the Cystic  Fibrosis (CF) Gene An autosomal recessive disorder 1. Chronic bronchopulmonary infection 2. Bronchiectasis 3. pancreatic insufficiency 4. biliary cirrhosis  5. meconium ileus in neonates 6. distal intestinal obstruction syndrome 7. male infertility  The disease was mapped by linkage  disequilibrium studies to  Chromosome 7q31 
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5 Chromosome Walking:  Cloning a contiguous DNA region using DNA probes of overlapping clones
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6 Positional Cloning 1. All genes in the interval are candidate genes for the disease. 1. Narrow down to THE GENE by examining spatial and temporal  distribution of gene expression.  1. Confirm by comparing individuals with disease to normal people in  relation to expression (mRNA) and DNA sequence.
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7 Positional Cloning of the Cystic Fibrosis (CF) Gene: Four   candidate genes There are four known genes in the  CF  gene region
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8 Positional Cloning of the Cystic Fibrosis (CF) Gene: One of the  candidate genes are confirmed  Northern Blot Analysis of  CF  Candidate Genes Of the 4 candidate genes, only one is expressed in the pancreas and  lungs, the tissues affected by cystic fibrosis.
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9 Positional Cloning of the Cystic Fibrosis  (CF) Gene The Cystic Fibrosis Gene Encodes a Cell Membrane Protein. That Regulates the Passage of Chloride Through the Cell Membrane. Expressed in the  lung and pancreas.
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10 Positional Cloning of the Cystic Fibrosis (CF) Gene: All genes in that DNA interval are  candidate genes
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11 Positional Cloning of the Cystic  Fibrosis (CF) Gene DNA sequencing of the  CFTR  gene reveal that all Cystic Fibrosis Patients  Have a Mutation in the  CFTR  Gene- GENE CONFIRMED But many different mutations in CFTR can cause cystic fibrosis in different  individuals! (Allelic heterogeneity)
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12 Cloning Alkaptonuria Gene Based on Cross-Species Homology
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13 Cloning Alkaptonuria gene based on  Cross-Species Homology
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14 1. Construction of cDNA libraries 2. Determine DNA sequence of individual clones 3. Submit sequence to Genbank at the National Center for 
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Lecture 20 Chapter 20 - PowerPoint Slides for Introduction...

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