1. Genetic Engineering
    Process of changing the genetic make up of a living organism or cell by artificial means
  2. 4 Basic Tools in Genetic Engineering
    • Restriction Enzymes
    • DNA Ligase
    • Vectors
    • Host Organisms
  3. Restriction Enzymes(endonucleases)
    • Found in bacterial cells
    • Function: Digest DNA molecules into restriction fragment
    • Natural Function: Protect bacteria from foreign DNA, as enzymes destroy foreign DNA
    • Bacterial DNA has methyl groups added to Adenine or Cytosine nucleotides in restriction site prevent digestion
  4. Restriction Enzymes
    • Very specific
    • Recognize and bind to short nucleotide sequences call restriction sites
    • Palindromic
    • Enzymes binds to restriction site and breaks covalent phosphodiester bonds at specific points in both strands
  5. Sticky ends
    • staggered cut with single stranded ends
    • These ends can anneal together by forming Hydrogen bonds to complementary sticky ends from other DNA cut up by SAME enzyme
  6. Blunt ends
    Simple cut across both stands at single point
  7. DNA Ligase
    Enzyme that seals nicks present in DNA molecule - catalysing formation of PHOSPHODIESTER BONDS between adjacent nucleotides in DNA molecule
  8. Vectors
    DNA molecule which carries target DNA into host cell
  9. Cloning Vector
    A vector that carries target DNA into host cell and allows foreign DNA to be reproduced in large quantities in host cell
  10. Properties of cloning vectors
    • Must be able to enter and remain in the host cell
    • Must contain an origin of replication
    • Contain marker gene that will give visible phenotype to bacteria that is easily detected
  11. Host organisms
    Cells in which recombinant DNA can be replicated or recombinant protein can be produced
  12. Problems of cloning Eukaryotic Gene using Bacteria as eukaryotic genes and products are processed differently from those of prokaryotes
    • 1. Eukaryotic genes contains introns while prokaryotic genes do not. Bacteria are unable to excise introns from RNA
    • Eukaryotic introns will be used in translation, causing wrong amino acids to be incorporated into the protein
    • Intron contains a stop codon that leads to premature termination of translation
    • Protein is not functional
  13. Solution
    • Use mRNA of the gene as introns would have been removed from the mRNA
    • Use enzyme convert RNA to DNA then insert DNA into a vector as RNA unstable
  14. Problem: mRNA transcribed from eukaryotic gene may not be translatable on bacterial ribosomes
    Use a eukaryotic host cell
  15. Problem: Protein must be modified after translation, bacteria no cellular machinery (RER/Golgi) for this
    Use eukaryotic host cell
    RNA is used as a template to make a complementary strand of DNA (cDNA)
  17. Process of Reverse Transcription
    • Isolate mRNA of desired gene from appropriate cell
    • Add enzyme reverse transcriptase which will use the mRNA as a template to make a cDNA strand
    • Add RNaseH to degrade the mRNA strand
    • Add DNA Polymerase which will use the cDNA to synthesise 2nd strand of DNA
    • Double stranded cDNA produced (inserted into plasmid vector)
  18. Procedure to clone eukaryotic gene using bacteria to produce eukaryotic protein
    • 1. Isolation of plasmid DNA and eukaryotic DNA (cDNA of gene of interest)
    • 2. Insertion of eukaryotic cDNA into plasmids
    • 3. Insertion of recombinant plasmids into bacteria (Transformation)
    • 4. Selection and cloning of transformed cells with recombinant plasmids
    • 5. Identification of bacterial colonies carrying gene of interest - gene probes
    • 6. Mass production of protein of interest
  19. 1. Isolation of plasmid DNA and eukaryotic DNA
    • Plasmid obtained from E.Coli
    • 2 marker gene
    • ampR - ampicillin resistance
    • lacZ - codes for enzyme beta-galactosidase (lactose breakdown)
    • Plasmid restriction site lies within lacZ gene
  20. 1. Isolation of plasmid DNA and eukaryotic DNA
    • Human or Eukaryotic DNA
    • Insulin mRNA extracted from pancreatic cells
    • Reverse transcriptase added to insulin mRNA
    • Uses the mRNA as a template to form cDNA
    • cDNA has blunt ends - sticky ends need to be attached to it
    • DNA sequences (oligonucleotide linkers) containing appropriate restriction site are ligated to the ends of cDNA using DNA ligase
    • Linkers will be cut by restriction enzyme to produce sticky ends
  21. 2. Insertion of human DNA into plasmids
    • i) Bacterial plasmid and cDNA of insulin gene digested with same restriction enzyme to produce complementary sticky ends (Restriction enzyme cuts plasmid DNA at single restriction site, disrupts the lacZ gene)
    • ii) cDNA of insulin gene and disgested plasmids are mixed to allow sticky ends of DNA from both sources to anneal by complementary base pairing
    • iii) Mixture incubated with DNA ligase
    • annealed strands joined covalently by phosphodiester bonds - recombinant plasmids
  22. Ligation mixture
    • Human cDNA fragments that have annealed to themselves
    • Plasmids DNA that has reannealed themselves
    • Recombinant plasmid
  23. 3. Transformation (high inefficient only about 1%)
    • Electroporation - cells are subjected to brief electric shock.
    • This makes pores appear transiently in the cell membranes of the BACTERIAL cells, making the membranes permeable to DNA and allowing the DNA molecules to enter the cells
  24. 4. Selection & cloning of transformed cells with recombinant plasmids
    Transformed cells plated onto nutrient agar plates containing ampicillin and sugar called X-gal (incubate at 37°C)

    Untransformed cells and bacteria transformed by cDNA fragment won't grow cos' no ampR gene

    Hence only reannealed or recombinant will grow
  25. 4. Selection & cloning of transformed cells with recombinant plasmids
    Recombinant plasmid - insertion of cDNA into plasmid within lacZ gene has disruped lacZ gene

    • Hence enzyme β-galactosidase not produced
    • X-gal not hydrolysed
    • Bacterial colony appear white (recombinant plasmid)
    • Pick these colonies

    Bacterial colonies carrying reannealed plasmid will synthesise β-galactosidase, catalyse hydrolysIs of X-Gal to produce BLUE PIGMENT
  26. 5. Identification, verification of recombinant bacteria carrying gene of interest using gene probes
    • Gene probe: short sequence of DNA designed to be complementary to part unique of the gene of interest
    • Nucleotides radioactively labeled
    • Binds to target sequence by complementary base pairing, tagging the gene of interest
    • Detected via autoradiography - involves exposing sample to X ray film
    • Radiation blackens the film, show location of radioactively labeled substances
  27. Process
    • White colonies identified grown on agar plate
    • Transfer to filter
    • DNA denatured with chemicals to separate 2 strands
    • Radioactive DNA probe (single stranded DNA) complementary to portion of gene of interest is then added to filter
    • Anneal to gene of interest by H bonds
    • Perform autoradiography on filter
    • Black spots formed on film show positions of where probe has bound to gene of interest
    • Base on location of black spots
    • Go back to master plate
    • Pick colonies that have gene of interest inserted in plasmids
  28. 6. Mass Production of protein of interest
    • Bacterial colonies identified to carry gene of interest picked and grown in large quantities in suitable medium.
    • Hormone produced extracted, purified and packaged.
  29. Advantages of Protein Production Using Bacteria
    • Bacteria reproduce very rapidly - large amounts of protein can be produced quickly
    • Able to produce human proteins identical to those in our bodies - protein will not stimulate immune response
    • Eliminates risk of zoonosis
    • Overcomes ethical objections of using live animals for production of proteins
  30. DNA Libraries
    • Genomic Library
    • cDNA Library
  31. Genomic Library - collection of clones containing fragments of an entire genome (both coding and non-coding DNA)
    • Human DNA cut into fragments using restriction enzymes
    • Fragments each ligated to vector DNA (plasmids)
    • Plasmids transformed into bacteria which are then plated on an agar culture plate
    • Transformed bacterium grows separately on the plates and forms bacterial colony
    • Each colony contains only 1 recombinant DNA molecule
    • Collection of millions of these colonies constitutes the clone library
    • Provided sufficient colonies are generated, any human genomic sequence should be represented in at least 1 colony
  32. cDNA Library - expressed genes (extrons)
    Subset of genomic library
    No non coding DNA
    Only those transcribed
    • DNA copies of expressed mRNAs are obtained
    • Double stranded DNA copies - cDNA
    • i) mRNA transcripts are reversed transcribed into double stranded cDNA using reverse transcriptase
    • Make complementary DNA strands using DNA polymerase
    • ii) cDNA inserted into vectors
    • iii) Plasmids transformed into bacteria, plated on agar culture plate
    • iv) Each transformed bacterium grows separately on plates and forms bacterial colony
    • Each colony only contains 1 remcombinant cDNA molecule
    • v) collection of millions of these colonies makes up the cDNA library
    • Provided sufficient colonies are generated, any human mRNA sequence should be represented in at least 1 colony
  33. Differences between genomic library and cDNA library
    • Genomic Library: Contains both coding and non coding sequence
    • Same content from all cells of one organism
    • Contains only 1 version
    • cDNA Library: Contains only coding sequence
    • May differ depend on stage or type of cell due to differential gene expression
    • contain multiple versions of 1 gene due to alternative splicing and multiple copies due to greater gene expression
  34. Polymerase Chain Reaction
    • Any specific sequence of DNA can be quickly amplified without cells
    • Reduce impurities
  35. PCR
    • DNA incubated in test tube with
    • 1.DNA POLYMERASE (taq polymerase)
    • 2.supply of deoxyribonucleotides
    • 3. 2 DNA primer sequences (forward and reverse) [short pieces of single stranded DNA specially designed to flank DNA sequence to be amplified
    • Denaturation 95°C
    • Excess forward and reverse primer mixed with DNA fragment to be amplified
    • To ensure that when temp decrease, the DNA would not reanneal
    • Mixture of primer and fragment heated to 95°C to break the hydrogen bonds between bases of double stranded DNA fragment, separating it into single strands
  37. PCR - Annealing of primers
    • Solution cooled to 55°C
    • Primers will base pair with complementary sequence in DNA to be amplified
  38. PCR - Primer Extension
    • 72°C
    • Heat stable Taq Polymerase use primers to synthesise new DNA strands
    • Nucleotides added to 3' ends of both primers and are complementrary to DNA template
    • As both DNA strands replicated, there are now two copies of the original fragment
  39. Each cycle 2 copies
    After 2 cycle?
    After 3 cycle?
    • 22 = 4
    • 23 = 8
  40. Advantages of PCR
    • Produces large numbers of copies of DNA in a short span of time
    • Cell free method requires no clean up of unwanted cellular debris
    • Efficient amplification of targets up to 35kilobases in length
    • Specific process that targets only desired DNA, starting material does not have to be purified DNA as primers are designed to flank the sequence to be amplified
  41. Applications of PCR
    • DNA fingerprinting in criminal investigations and paternity tests (starting pt is very little, so must amplify to investigate)
    • Detecting genetic defects in very early embryos
    • Disease detection
    • Genetic tests - mutation detection
    • Palaeontology - amplification of ancient DNA fragments from fossils
    • Classification of organisms - molecular homology (amplify certain sections of DNA to compare with other organisms' DNA)
  42. Limitations of PCR
    • Must know nucleotide sequence of at least one short DNA sequence on each side of region of interest in order to synthesise PCR primers
    • ERRORS
    • Taq polymerase no proofreading capability - produce higher than normal frequency of replication errors
    • Use Pfu Polymerase/Vent Polymerase
  43. Gel Electrophoresis
    • Separation of macromolecules - nucleic acid/proteins
    • Nucleic acids separated on basis of rate of movement through gel in electrical field
    • Rate of movement depends on size, shape and electrical charge
  44. Gel electrophoresis
    • Tells us how many TYPES of DNA fragments/proteins present
    • Tells us size of DNA fragments/proteins (deduce identity of fragment)
    • Allows purification of specific fragment of DNA/specific protein from mixture
  45. Gel Electrophoresis
    Involves use of agarose & acrylamide gels acts as molecular sieves, retarding passage of large molecules more than small molecules

    • Agarose : Better sieves for large molecules
    • Acrylamide Gels : Separate small DNA molecules

    DNA relatively constant charge per unit mass and linear shape. Separated entirely based on size
  46. Principles of Gel Electrophoresis
    • Gel prepared by pouring melted agarose into container
    • Agarose solidifies, gel matrix forms consist of long tangled chains of agarose
    • Mixture of DNA fragments to be separated is layered in a well at the top of gel
    • DNA -vely charge due to phosphate grps
    • MOVE TOWARDS +VE ELECTRODE when electric field applied
    • Molecules move through pores in gel at rate inversely proportional to their chain length
    • Shorter faster hence further in fixed amount of time
  47. GE
    • Ethidium bromide is a fluroscent dye that binds to DNA and is added to the gel so that DNA can be visualized
    • DNA with EB will show up under UV light

    • Shorter bands found nearer to ANODE (+ve)
    • Longer bands found nearer to CATHODE (-ve)

    Some bands appear bright because they contain more DNA material and hence trap more EB
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