Lecture 13-18-2011-summer

Lecture 13-18-2011-summer - Deconstructing Genome Digital...

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Unformatted text preview: Deconstructing Genome Digital analysis of Genome Conserved gene function • Some examples are surprising. • Eye formation: EYELESS mutant • Gene is required for eye development. • Similar gene is required for eye development in mammals. PAX5 What does genetics tell us about life? • All living things use DNA for storing biological information (with the exception of some viruses). • Suggests a common ancestor • Same genetic code • Distantly related organisms contain genes with similar sequences. Cytochrome C Cloning and hybridization • After years of research in mice, scientists have identified a gene (BND, not a real gene) that causes blindness when mutated. • Your job now is to: – Isolate this gene from humans. – Determine if similar mutations are responsible for blindness in humans. Cloning BND from humans • What does it mean to clone? Cloning BND from humans • What does it mean to clone? Cloning BND from humans • What does it mean to clone? • Scientist use the word to describe two related but different procedures: • 1. vt to produce an organism that is genetically identical to its parent, by vegetative reproduction or a laboratory technique, or be produced in this way Cloning BND from humans • What does it mean to clone? Cloning BND from humans • What does it mean to clone? • 2. vt to produce an exact or near copy of an object or product • Produce copies of a particular DNA fragment. Fragment of interest Vector Cloning BND • Three approaches: • Genomic library-Collection of cloned genomic DNA fragments. • cDNA library-Collection of cloned cDNA fragments. cDNAs are DNA copies of mRNA molecules produced by reverse transcription. • PCR-Polymerase chain reaction Cloning BND: Genomic library • Cloning the BND gene from genomic DNA is a challenge. • Human genome has 3X10 9 bp and 25,000 genes. • How do we find our gene? • Overview: – Break up the genome into fragments. – Clone all of the fragments. – Identify which of the clones contains the BND gene. Breaking up the genomic DNA • Can be done mechanically. • Usually done with restriction enzymes, which cleave DNA at specific sequences. • Blunt or sticky ends. • Which restriction enzyme do you use? • What size fragments do you want? Restriction enzymes cut DNA molecules at specific locations and produce either blunt or sticky ends. The number of base pairs a restriction enzyme recognizes determines the average distance between sites and thus the size of fragments produced. • Probability that a four-base recognition site will be found in the genome = ¼ x ¼ x ¼ x ¼ = 1/256. Breaking up the genomic DNA • Sau3AI: – GATC – 4 4 =256bp • EcoRI – GAATTC – 4 6 =4,096bp • NotI – GCGGCCGC – 4 8 =65,536bp • Average size of a gene is 15kb (15,000bp) Breaking up the genomic DNA • Alternatively, you can partially digest with an enzyme like EcoRI or Sau3AI....
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This note was uploaded on 11/07/2011 for the course BIO 325 taught by Professor Saxena during the Summer '08 term at University of Texas.

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Lecture 13-18-2011-summer - Deconstructing Genome Digital...

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