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Genetics

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  1. Genetics BIO205 Dr. Todd Eckdahl Missouri Western State College

  2. Genetic Engineering

  3. Genetic Engineering: Overview • Recombinant DNA technology • Genetic engineering • Gene cloning

  4. Genetic Engineering: Applications • production of drugs and biochemicals • Genetically modified organisms • Analysis of genetic disease alleles • Correction of genetic defects

  5. DNA Cloning Flowchart • Recombinant DNA production • Genetic transformation • Selection • Screening

  6. Recombinant DNA production • Source DNA and vector DNA combined • Restriction enzymes • DNA ligase

  7. 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

  8. 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

  9. 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

  10. 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

  11. 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

  12. 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)

  13. Genetic transformation • Introduce recombinant DNA into host • Bacteria • Fungi • Plants • Animals • Human cells (gene therapy)

  14. 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

  15. 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

  16. 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

  17. 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

  18. Applied Genetic Engineering • Recombinant DNA and animal growth rate • Transgenic animals with growth hormone gene • Control of highly active promoter

  19. 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

  20. 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

  21. 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

  22. 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

  23. Functional Genomics • Patterns and mechanisms of gene expression focused on genome-wide patterns • 2 DNA chips: oligonucleotides and denatured, double-stranded DNA sequences