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  1. 11 Genetic Engineering and Functional Genomics

  2. Genetic Engineering: Overview Methods of genetic manipulation are termed: • Recombinant DNA technology • Genetic engineering • Gene cloning Applications include: • Isolation of specific genes • Production of specific proteins

  3. Genetic Engineering: Overview • Increased efficiency in production of drugs and biochemicals • Generation of organisms such as plants with desired traits • Analysis of genetic disease alleles • Correction of genetic defects

  4. Restriction Enzymes • Restriction enzymes cut double-strand DNA at specific recognition sequences which are 4-6 base pair palindromes = 5’-3’ sequence is identical on both DNA strands • Many restriction enzymes cut the two DNA strands at different points which generates complementary single-strand ends = sticky ends

  5. Restriction Enzymes • Sticky ends formed by restriction enzymes permit circularization of the DNA restriction fragment by complementary base pairing • Some restriction enzymes cut at the same point in the two DNA strands which generates blunt end DNA fragments

  6. DNA Cloning • Vector = DNA molecule which can be used to amplify gene sequences • Gene cloning = the insertion of genetic material into a vector in order to isolate specific genes • Cloning methods involve the cleavage of insert and vector DNA with the same restriction enzyme to generate complementary sticky ends

  7. DNA Cloning

  8. DNA Cloning: Vectors Properties of useful vectors: • Vector DNA can be introduced into a host cell • Vector contains a replication origin so it can replicate inside a host cell • Host cells containing vector can be readily identified due to presence of antibiotic resistance gene or other selectable marker

  9. pBluescript II: modern vector

  10. DNA Cloning: Vectors Cloning vectors used with E. coli: • 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

  11. DNA Cloning: Vectors

  12. 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

  13. DNA Cloning: Vectors

  14. Genetic Engineering • Gene Cloning • Any gene can be isolated and purified • Recombinant DNA • Cloned genes can be altered in any way • Many methods: PCR; oligos, chemicals, etc. • Genetic Transformation • Altered genes can placed into (any) organism • Many methods: chemicals, electroporation gene guns, etc.

  15. 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)

  16. cDNA Cloning • Insert DNAs to be cloned can be generated from mRNAs using the enzyme reverse transcriptase • Reverse transcriptase generates a double-strand copy of the mRNA =cDNA which is ligated to vector DNA • mRNAs are obtained from cells producing protein encoded by targeted gene

  17. cDNA cloning

  18. Recombinant DNA: Screening • Colony hybridization is used to identify bacterial colonies containing the gene of interest • Bacterial transformants are detected by antibiotic resistant phenotype • Colonies are transferred to filter and probed with labeled DNA homologous to gene to be cloned

  19. Colony Hybridization

  20. Gene Cloning • Positional cloning or map-based cloning involves a determination of the chromosomal location of cloned DNAs relative to meiotic markers • Reverse Genetics involves site-directed mutagenesis or the insertion of mutations at targeted sites of cloned genes to identify the functional domains of specific genes

  21. 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

  22. Germ-Line Transfomation • Germ-line transformation in mice involves the insertion of genes into embryonic stem cells (from black strain) • Genetically altered cells are then inserted into embryo (white strain) • Offspring are mosaics; if cells from black strain enter germline, offspring of mosaics are black

  23. Germ-Line Transfomation

  24. 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

  25. Gene Targeting

  26. 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

  27. Transformation Rescue • Determine experimentally the physical limits of the gene • No general method to identify regulatory sequences • Ability of DNA fragment to correct genetic defect in mutant organism

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

  29. 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

  30. 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

  31. Genome Analysis • Recombinant DNA methods can be used to physically map genomes and determine DNA sequence • Euk. Genomic size is in range of 10 million base pairs to 10 billion base pairs • Large fragment DNAs can be produced by restriction enzymes and analyzed or isolated by electrophoresis

  32. Large-Scale DNA Sequencing • Human Genome Project involved a determination of the DNA sequence of the human genome • The complete sequence of the E. coli genome is known • The yeast genome was the first eukaryotic genome sequenced • Large-scale sequencing requires highly automated methods

  33. Large-Scale DNA Sequencing • Over 60 bacterial genomes sequenced • Biases in genomes chosen for sequencing

  34. Eukaryotic Sequencing • Reveals fewer genes than expected • 32,000 in Homo sapiens • Comparable breakdown of genes for cellular/transcriptional/metabolic processes in humans and flies • Functional genes of greater complexity in vertebrates

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