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Lecture 5

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University of Toronto Mississauga
Robert Gerlai

Lecture 5 - October 19, 2012  ggene cloning single copy of gene placed in bacterium which replicates  The point of cloning is to create a large number of genes that makes it easier to analyze and manipulate  A single gene is just too small to manipulate  Special restriction enzyme that cut out particular genes to add to a plasmid (circular gene, vector that carries gene)  This process called recombinant DNA technology  Technology first utuilized 30 years ago in human medicine o Human Insulin first recombinant protein created o Before the only insulin available for human use came from pigs  Now this method is used for testing effects of genes  cDNA = complementary DNA, based upon mRNA (not have introns, only has genes relevent to creating proteins etc [just coding region] therefore is much smaller and compacted)  Study gene expression mostly by analyzing mRNA levels in a sample based on hybridization  Microarray/DNA chip small piece of glass (few square cm) that has 40000genes*12 pieces of DNA (probes) incl. Can map entire genome with it.  Other types of studying incl northern blot (most common) and quantitative PCR o Reverse transcriptase Polymerase chain reaction (RT PCR) o mRNA purified (taken out of tissue sample) and RT used to turn mRNA into cDNA and DNA o This is done b/c DNA is very stabe vs RNA o DNA is then amplified by making lots of copies thru PCR (amplification procedure similar to cloning)  Each oligonucleotide probe (there can be thousands) on a microarray is capable of detecting many hybridizations o Probe has synthetic DNA that allows detection of hybridization  Light signal means complementary sequence is present  The closer the animal is to humans genetically the more relevant the discoveries will be for humans  However even distantly related species can still be relevant in human medicine discoveries  Fish have. 70% gene sequence homology with humans  Knock out = genes inactivated by a directed mutation  Knock in = gene introduced in a specific locus to replace another gene  Transgenes = gene instoduced somewhere at random in the genome MUTATIONS  Germ-line mutations affect sperm/egg and can be inherited, somatic mutations are not passed on to offspring  Small scale mutations: substituion, deletion, insertion which all occur at the nucleotide bases, large scale mutation = translocation o Missense mutations= sin
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