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Lecture 5

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Biochemistry 2280A
Christopher Brandl

Recombinant DNA Technology Definition: The techniques which allow DNAfragments from different source to be recombined to make new molecules with unique features Synthetic Biology ● The construction of new biological parts and systems ● The redesign of existing, natural systems for useful purposes Comparison of classical genetic techniques and RDT: Classical Genetics Recombinant DNATechnology Slow: limited by the breeding time of the Rapid: as quick as a few days in some organism and chance genetic events organisms Limited to breeding species; must cross No limitations between the same species Significance of Recombinant DNA Technology  ● Research ● Biotechnology Biotechnology: ● Definition: the use of organisms to do work for man ○ modern biotechnology impacts many aspects of society and relies heavily on recombinant DNAtechnology Medicine ● Drug production and design rely heavily on recombinant DNAtechnology ● Diagnosis of disease - detect pathogens and disease causing genes through their DNA signatures ● Genetic counselling - does an individual carry a disease related allele Potential for gene therapy ● Many diseases result from having a defective gene ○ we can add back functional genes to cure diseases ■ these approaches can work, but often putting the gene back can cause other problems Agriculture ● Production of crops with unique features ○ Vitamin Aenhanced rice ○ Cold and drought resistant crops ○ Pest resistant corn Production of Novel Molecules in plants ● Large scale production of therapeutic drugs Manufacturing ● Novel products ○ spider silk ● More efficiently ○ detergents Environment (bioremediation) ● Engineer a bacteria that can eat oil, PCBs, styrofoam etc. Forensics and Law  ● DNAfingerprinting - CSI Recombinant DNA Technology Case Study ● You have identified a human protein that will be of tremendous value in treating disease (HGH) ● Your goal is to mass produce this protein (YFP - Your Favourite Protein) Why is Expression of Your Favourite Protein using Recombinant DNATechnology Important? ● Many proteins are difficult to obtain from their native source. Recombinant DNA technology can dramatically increase expression and facilitate purification The process of expressing YFP will Require: ● Cloning YFG ● Introducing YFG into E. coli ○ E. coli are most commonly used to express YFP due to the following reasons: ■ grow and reproduce quickly and inexpensively ■ protein extracts are made easily ■ genetic engineering is well worked out and is simple; techniques are well established ■ multicopy plasmids and strong promoters can drive expression ● Purifying the protein from E. coli The first issue we need to think about: ● We need a source of the gene, should it be genomic DNAor mRNA? ● It should be DNAonly for the few rare genes that lack introns ● Introns are a problem in expression of eukaryotic genes in E.coli as bacteria lack introns thus have no mechanism to remove them Cloning YFG ● To clone YFG, we will use mRNA(cDNA) ○ this is because by definition, mRNA (cDNA) does not contain introns, and that each mRNA(cDNA) encodes a single gene ● Do all tissues express YFG as mRNA? No. ● How do we find a tissue or human cell line that expresses a lot of YFG? ○ Use hybridization and observe on a Northern blot Nucleic Acid Hybridization ● Denaturation/melting: heating (or OH) double stranded DNAresults in the molecule breaking down ● Renaturatio
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