Gene Technology

• Recombinant DNA technology:

  1. Isolation of genes
  2. Insertion
  3. Transformation
  4. Identification
  5. Growth/cloning
• Ways to produce DNA fragments:

  • Conversion of mRNA to complementary DNA using reverse transcriptase
  • Restriction enzymes cut a fragment containing the desired gene from DNA
  • Creating gene in the gene machine
• Why have sticky ends:

  • Joining two pieces of DNA
  • By complementary base pairing
• Blunt ends used for:

  • Polymerase chain reaction (make more of gene)
  • Separation by gel electrophoresis
• Isolation - reverse transcriptase:

  • Makes cDNA from mRNA templates
  • Free DNA nucleotides bind to single-stranded mRNA
  • Reverse transcriptase joins DNA nucleotides together to form single-stranded cDNA
  • DNA polymerase forms double-stranded cDNA
• Advantages of using mRNA:

  • mRNA is easier to obtain from cytoplasm
  • Introns already removed
  • Cells contain a maximum of two alleles
• Isolation - restriction endonuclease:

  • These enzymes hydrolyse DNA at specific recognition (base) sequences
  • Different restriction endonucleases hydrolysed DNA at different specific recognition sequences by complementary shape
  • Recognition sequences are palindromic
• If the recognition sequence for the selected restriction endonuclease occurs within the DNA fragment isolated, this will cut the gene and it will not code for a functional protein
• When a DNA fragment is isolated a promoter region can be added to allow expression of the gene. A terminator region can be added to stop the transcription of the gene.
• Isolation - gene machine:

  • Desired nucleotide sequence fed into a computer
  • Synthesis of oligonucleotides (short nucleotide sequence)
  • Assembly of gene (nucleotides overlapped and joined using PCR)
  • Gene inserted into bacterial plasmid
• Benefit of the gene machine:

  Protein structure -> DNA = DNA without introns

  Easily transcribed and translated by prokaryotes as no introns present
• Insertion of gene:

  • Vector DNA cut using the SAME restriction endonuclease used to isolate the DNA fragment (isolation)
  • Complementary sticky ends produced between ends of DNA fragment and vector DNA
  • Target DNA fragment anneals to vector DNA by complementary base pairing of sticky ends
  • DNA ligase is used to join DNA fragments and vector DNA - forms phosphodiester bond
  • Forms recombinant DNA
• Transformation - The recombinant DNA vector is transferred into a host cell (bacteria)
• Outline a method for in vivo gene cloning:

  • Cut desired gene from DNA of desired organism
  • Using restriction endonucleases
  • make artificial DNA with correct base sequence
  • Using DNA polymerase
  • Cut plasmid open
  • Same restriction endonuclease
  • Complementary stick ends on desired DNA and vector DNA joined by DNA ligase
  • Transferred into bacterial cells
• How changed plasmids enter bacteria:

  • Heat shock OR electric current
  • Creates temporary pores in cell membrane
  • Plasmids enter through pores
• Identification of host cells:

  • Not all vectors take up the target DNA to become recombinant
  • Not all host cells become transformed by taking up recombinant vectors
  • Scientists only want transformed host cells so will add a marker to identify these (e.g. fluorescence)
• Market gene - Allows easy identification of cells that have taken up a genetically transformed plasmid
• Types of gene markers:

  • Antibiotic resistance gene (only transformed organisms grow if resistant)
  • Fluorescence gene (easy to identify using UV light)
  • Enzyme markers (lactase turns colourless substrates blue)
• Identification - antibiotic resistance genes:

  • Some cells will not take up any plasmid - killed by both types of antibiotic
  • Some cells will take up the original plasmid - resistant to both types of antibiotic
  • Some cells taking up the transformed plasmid will be resistant to only one type of antibiotic but not the other
• Cloning:

  • Separation
  • Annealing
  • Synthesis
• Extremophile - A microorganism that grows in extreme conditions.
• Process of PCR:

  • Heat DNA to 95 degrees to break hydrogen bonds and strands separate
  • Add primers and add nucleotides
  • Cool to 50 degrees allows binding of nucleotides or primers
  • Add heat stable DNA polymerase
  • Heat to 75 degrees
  • DNA polymerase joins nucleotides together
  • Repeat cycle many times
• Primers - Short pieces of single stranded DNA with complementary base sequences to bases at the start of the DNA fragment. Prevents DNA strands from sticking together and allows DNA polymerase to attach and joins free nucleotides together
• In vivo cloning:

  • Used to produce protein or mRNA from inserted DNA + target DNA
  • Cells have mechanisms for correcting errors
  • Can be used to clone large DNA fragments
  • Slow - one DNA replication per division
  • DNA/RNA produced is spliced to remove introns
  • Need to isolate DNA fragments from DNA before insertion
  • Cant copy partly broken down DNA
  • Need large sample of DNA to start with
• In vitro cloning:

  • Only be used to copy DNA
  • No error prevention mechanisms so error rate is high
  • Unreliable when cloning longer DNA fragments
  • Fast - millions copies made in hours
  • No modification of DNA (not spliced)
  • Only replicated target DNA
  • Can be used to copy partly broken DNA
  • Only small sample of DNA needed at start
• DNA graph explanation:

  • Less strands at beginning so slow increase
  • DNA strands double each cycle so large range in numbers (expontential increase)
• Why DNA graph plateaus:

  • Less DNA nucleotides left so less DNA strands made
  • Less primers present so less DNA strands made
• Why some organisms contain desired gene in all cells:

  • Plasmid DNA in organism DNA
  • Semi-conservative replication occurs
  • Mitosis of cells allows DNA to be copied for all cells
• Benefits of recombinant DNA technology:

  • Develop medical applications (insulin)
  • Develop agricultural applications
  • Better understanding of biological processes
  • Design new or improve industrial processes
• Concerns of recombinant DNA technology:

  • Antibiotic resistance spread if resistance marker used
  • Inserting new genes may alter current genes leading to toxic plants etc
  • Large corporations may have large hold over technology and create a monopoly
• Recombinant DNA technology can be used to insert a gene for a functioning protein into the cells of people suffering genetic disease
• As plasmids cannot carry DNA into human cells, viruses or liposomes (lipid-soluble) are used as vectors in gene therapy
• Using viruses as vectors are safe because they only contain the desired material so does not damage the cell
• Eugenics - manipulation of genetic properties of a population for racial improvement
• Somatic gene therapy - DNA transferred to our normal body tissues
• Germ line gene therapy - DNA transfer to cells that produce eggs or sperm
• Germ line therapy ethics:

  • Further defects in embryo may occur despite successful gene transfer
  • Modified individual did not have a say for their genetics to be modified
  • Can suppress unfavourable characteristics and enhance favourable ones
• Somatic gene therapy:

  • Not all cells take up new DNA
  • Not all cells express DNA allele
  • Only some tissue types can be permanently fixed
  • Multiple treatments needed
  • Body can produce immune response to vector
• Gene probe - single stranded DNA molecule with complementary bases sequence
• Why exons used in DNA probing:

  • Introns do not code for an amino acid sequence
  • Mutations of exons affect primary and tertiary structure
  • Important to know whether exons are affected for offspring