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Genetics. BIO205 Dr. Todd Eckdahl Missouri Western State College. Genetic Engineering. Genetic Engineering: Overview. Recombinant DNA technology Genetic engineering Gene cloning. Genetic Engineering: Applications. production of drugs and biochemicals Genetically modified organisms

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Genetics

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Genetics

BIO205

Dr. Todd Eckdahl

Missouri Western State College


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Genetic Engineering


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Genetic Engineering: Overview

  • Recombinant DNA technology

  • Genetic engineering

  • Gene cloning


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Genetic Engineering: Applications

  • production of drugs and biochemicals

  • Genetically modified organisms

  • Analysis of genetic disease alleles

  • Correction of genetic defects


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DNA Cloning Flowchart

  • Recombinant DNA production

  • Genetic transformation

  • Selection

  • Screening


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Recombinant DNA production

  • Source DNA and vector DNA combined

    • Restriction enzymes

    • DNA ligase


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Restriction enzymes

  • Restriction enzymes

    • cut double-strand DNA

    • recognition sequences are 4-6 base pair palindromes

  • Many restriction enzymes cut the two DNA strands at different points

    • generates complementary single-strand ends, or sticky ends


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DNA Cloning: Vectors

Properties of useful vectors:

  • can be introduced into a host cell

  • contain a replication origin

  • host cells can be identified due to presence of selectable marker


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DNA Cloning: Vectors

  • Plasmid: insert DNA = 5 kb; autonomous replication; contains antibiotic resistance genes

  • Bacteriophage lambda: insert = 15 kb; recombinant DNA packaged into phage particles used to infect E. coli


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DNA Cloning: Vectors

  • Cosmid: insert = 40 kb; combination of plasmid and phage vectors which can replicate as plasmids and are packaged into phage particles to infect E. coli

  • P1 phage: insert = 85 kb; useful for cloning large DNA fragments


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Recombinant DNA

  • Construction of recombinant DNA molecules may involve cleavage of both vector and insert DNA with the same restriction enzyme in order to generate complementary sticky ends

  • DNAs cut with the same restriction enzyme base pair if mixed together

  • DNA ligase covalently links the DNAs


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Genome Analysis

Three classes of artificialchromosomes are used as vectors for large DNA fragments:

  • P1 artificial chromosomes(PACs)

  • bacterial artificial chromosomes(BACs)

  • yeast artificial chromosomes(YACs)


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Genetic transformation

  • Introduce recombinant DNA into host

    • Bacteria

    • Fungi

    • Plants

    • Animals

    • Human cells (gene therapy)


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Selection and Screening

  • Host cells that receive vector can be selected for

    • Eg. Antibiotic resistance in bacteria

  • Host cell containing clone of interest must be “screened for”

    • Relies on information

    • Eg. Gene – colony lift with DNA probe

    • Eg. Protein – colony lift with antibody probe


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Germ-Line Transformation

  • Germ-line transformation involves the insertion of genes into the reproductive cells of an organism which permanently alters the genetic content of the individual and all offspring = transgenic animals

  • Transgenic animals are used to study the functions of specific genes in development or disease processes


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Gene Targeting

  • Gene targeting in embryonic stem cells involves homologous recombination between target gene in vector and target gene in genome

  • Target gene in vector contains unrelated DNA so that recombination disrupts function of targeted gene

  • Transgenic mice have mutant gene


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Alteration of Plant Genomes

  • Recombinant DNA can also be introduced into plant genomes

  • Gene transfer procedure uses Ti plasmid of Agrobacterium tumefaciens

  • Inserted genes replace portion of plasmid and a selectable marker is used to assess successful gene transfer


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Applied Genetic Engineering

  • Recombinant DNA and animal growth rate

  • Transgenic animals with growth hormone gene

  • Control of highly active promoter


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Applied Genetic Engineering

  • Agricultural crop plants are primary targets of genetic engineering to increase yield, hardiness and disease resistance

  • Annual growth rate can be genetically engineered

  • Engineered microbes can help degrade toxic waste


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Biomedical Applications

  • Recombinant DNA technology is used to produce large amounts of medically important proteins

  • Animal viruses such as retroviruses may prove useful vectors for gene therapy to treat single gene disorders

  • Recombinant DNA probes detect mutant genes in hereditary disease


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Genome Analysis

  • Recombinant DNA methods can be used to physically map genomes and determine DNA sequence

  • Genomic size is in range of 100 million base pairs

  • Large fragment DNAs can be produced by restriction enzymes and isolated by electrophoresis


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  • Human

    • 3.2 billion bp, ~22,000 genes

  • E. coli

    • 6 million bp, ~3200 genes

  • Yeast, S. cerevisiae

    • 12 million bp, ~6200 genes

  • Fly, Drosophila

    • 185 million bp, ~14,000 genes

  • Worm, C. elegans

    • 97 million bp, ~18,000 genes

  • Weed, Arabidopsis

    • 100 million bp, ~25,000 genes

  • Rice, Oryza sativa

    • 430 million bp, ~40,000 genes


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Functional Genomics

  • Patterns and mechanisms of gene expression focused on genome-wide patterns

  • 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences


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