geneticEngr2

geneticEngr2 - Genetic Engineering Techniques (Part 2)...

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Genetic Engineering Techniques (Part 2) Chapter 9 Deconstructing the Genome: DNA at High Resolution
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Whole genomic libraries contain all DNA in a genome cDNA libraries contain only information from gene’s exons Prepare total mRNA from a tissue Add reverse transcriptase and four deoxyribose nucleotide triphosphates, and primers to initiate synthesis
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cDNA library construction Fig. 9.10
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A comparison of genomic and cDNA libraries Fig. 9.11
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Advantage: cDNA copies do not contain introns or other non-coding material Disadvantage: Less efficiently transcribed genes are underrepresented LIBRARIES Advantage: A random sample of every sequence in the genome is represented Disadvantage: A lot of non-coding and repetitive sequences Genomic libraries cDNA libraries LIBRARIES OVERVIEW: LIBRARY – A COLLECTION OF DNA CLONES OBTAINED FROM ONE GENOME DONOR
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IDENTIFICATION OF A GENE IN A LIBRARY 1. Functional screening: By phenotypic complementation Yeast LEU2 E. coli leuB - mutant Leu + By protein product
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Expression vectors produce large amounts of specific polypeptide Vector contains promoter and regulatory sequences Vectors transformed into bacteria, yeast, or cultured mammalian cells Fig. 9.12 a
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Screening an expression library Expression library – entire cDNA library Probe library with fluorescently labeled antibody that binds to protein product of gene Fig. 9.12 b
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IDENTIFICATION OF A GENE IN A LIBRARY 1. Functional screening: By phenotypic complementation Yeast LEU2 E. coli leuB - mutant Leu + By protein product 2. Hybridization screening: By ability to hybridize with a homologous piece of DNA
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geneticEngr2 - Genetic Engineering Techniques (Part 2)...

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