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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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Dr. Todd Eckdahl

Missouri Western State College

genetic engineering overview
Genetic Engineering: Overview
  • Recombinant DNA technology
  • Genetic engineering
  • Gene cloning
genetic engineering applications
Genetic Engineering: Applications
  • production of drugs and biochemicals
  • Genetically modified organisms
  • Analysis of genetic disease alleles
  • Correction of genetic defects
dna cloning flowchart
DNA Cloning Flowchart
  • Recombinant DNA production
  • Genetic transformation
  • Selection
  • Screening
recombinant dna production
Recombinant DNA production
  • Source DNA and vector DNA combined
    • Restriction enzymes
    • DNA ligase
restriction enzymes
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
dna cloning vectors
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
dna cloning vectors10
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
dna cloning vectors11
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
recombinant dna
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
genome analysis
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)
genetic transformation
Genetic transformation
  • Introduce recombinant DNA into host
    • Bacteria
    • Fungi
    • Plants
    • Animals
    • Human cells (gene therapy)
selection and screening
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
germ line transformation
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
gene targeting
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
alteration of plant genomes
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
applied genetic engineering
Applied Genetic Engineering
  • Recombinant DNA and animal growth rate
  • Transgenic animals with growth hormone gene
  • Control of highly active promoter
applied genetic engineering22
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
biomedical applications
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
genome analysis25
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
    • 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
functional genomics
Functional Genomics
  • Patterns and mechanisms of gene expression focused on genome-wide patterns
  • 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences