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University of Toronto Mississauga
Craig Burkett

Test Monday 12 at PUMP Genetics • Isolate gene from rest of genome • Produce large enough • “cloning” • Ex using DNA fragment from bacteria o By using restriction enzymes to separate • Separate enzymes that allow site specific cutting of dna sequence • Vector – mini chrromose = transformation • As bacteria multlpies, vector is alos multiplied to poduct many copies o Imp: source of gen material! o Gene from dna nucleus contains still all non coding sequences  So it is large and difficult to analyze for function o Messenger RNA  Only coding sequence and axons so much simpler  Converted to dna first before cloning (complementary dna) o Imp to know sequence and compare to other sequences  Under what conditions is it expressed?  What functions in other organisms? o Expression studes how a gene is regulated in a tissue or cell type o Useful way : measuring levels of mrna from a particular gene in a particular tissue o Common technique --- northern blot o More sophisticated – (PCR) o Dna erase allows for gene expressions of 10,000’s of genes in 1 tissue in 1 experiment o Dna chips consis of thousand of dna probes attached to small surface of few sq cm’s o The mrna molecule are labelled fluorescently so they can be detected in the array o probes can hyperdize thousands of rnas o dna technology highlu effeceint for gene analysis o these experiments are useful to study environmental factors on gene expression o northern plot (only for one gene) vs micro erase (thousand of genes) o phaamrmogenomics –diffrential gene expression applied to drugs etc  may allow identification of new drug target,s evlaue action mech of a drug, or chose best candidate for optimal drug medicine, etc o toxigonemics – toxic substances effect n gene tissue o tissues  new ways to examine potential toxicity of a drug.  Which gene is increased or decreased by influence of a drug to udnerstad adverse effects of drugs and why  For liver toxicity in a rat for example (microaaray shows white line that shoes there is gene expression!)  IN CONTROL WE DONT SEE SAME THING o Model organisms are indespinsible tool to study genes o Yeast, bacteria, animals o Complex more – difficult more – but more relevant model vecoems to humans! o CLONING- genetically identical animal. Replicate of an animal.  Inbreeding – way it is done by lab  One new technique - nuclear transfer technology • Donor cell (sematic cell) • Somatic cell –diff from gamete cell! • Somatic cell – any cell taken from body that doesn’t have to do with reproduction • The nucleus is whetre teh dna is kept and that info is deployed • So procedure involve removing that nucleus and injecting to an infertiizlied immature egg • Site has its own info that will be discarded (osite) • Through stimulation allows cells to undergo mitosis and replicate • Once it is viable they inject in a surrogate mother • That’s how DOLLY was created • Very complicated procedure  A lot of controversy • Ethical considerations considered o Cloning in animal diff from gene cloning o Cloned animals are same as identical twins? o MITOCHODNRIAL DNA is diff (in twins it’s the same but clones have different) • Organsisms testing by inactiving the expression (or introduce a new gene) • MUTATION o Somatic (info not passed) vs germine cells (info is passed on) o Changes in dna sequence  May be large or small scale  Large – gain or loss of chromosomal rgions or switching if parts of chromosome (transolocations)  Small – nucleotide changes (substitutions, deletions or insertions) o Diff types of single cell mutations  Misense mutation • Change in nuclear type sequence which changes the amino acid sequence • Instead of glycen can be aspertine acid • So therefore changes protein acid and has effect on behaviorial level of the protein  Silent mutations • Change in the sequence but results in same amino acid so no chnge in protein  Non sense mutations • Immediate stop in amino acid sequence for some reason • In NOTES  Frame-shift mutations • If nucleotide inserted – the entire amino acid sequence will shift and protein and amino acid will be different as well • Mutations reduced by exposure b mutagens o Uv light o ionized radiatns such as x rays o Chemicals that can react with o r damage dna • Proofreading proliferiss – correct errors during dna replication, prevents deadly changes in dna sequence •
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