Pack 15 - Recombinant DNA technology

Pack 15 - Recombinant DNA technology

1. Producing DNA fragments

   • A gene is a section of DNA on a chromosome coding for one or more polypeptides and some forms of RNA
   • Recombinant is the combining of DNA together from two different organisms
   • Major ways fragments of DNA can be produced:
     • 1) conversion of mRNA to cDNA
     • 2) Restriction endonuclease enzymes cutting from mRNA
     • 3) Created in the "gene machine"
   • Process of GMO:
     • 1) Isolation of the gene of interest
     • 2) Insertion of gene
     • 3) Transformation, transfer gene and vector into host
     • 4) Identification of the host cells that have taken up the gene
     • 5) Growth/cloning of the host cells
   • It is difficult to transfer genes from eukaryotes into prokaryotes due to introns (non-coding DNA)
     • This can be overcome by using reverse transcriptase:
       • This is the formation of DNA using RNA template
         • Produces cDNA (no introns) which is:
           • Complimentary DNA
           • Made of DNA complementary to original mRNA
       • Process of making cDNA:
         • mRNA coding for a particular gene
           • mRNA acts as template which single stranded DNA, cDNA formed with reverse transcriptase
             • DNA is isolated by hydrolysis of RNA with an enzyme
               • Double stranded DNA is formed by incubating with DNA polymerase and DNA nucleotides
   • Using restriction endonucleases
     • These are a group of enzymes which cut the DNA at certain specific base sequences.
     • Recognition sequences are often 6 bp long and are palindromic
     • When cut, DNA can form one of two things:
       • sticky end - single stranded over hang
       • Blunt end - no single stranded overhang
         • Sticky ends are better than blunt ends because blunt end is non-specific and can go in the wrong way round
   • Making a gene in a gene machine:
     • 1) desired nucleotide sequence inputed into computer
       • 2) computer designs series of small overlapping DNA sequences
         • 3) Small single stranded sequences assembled by adding a nucleotide at a time
           • 3) join together to make gene
             • 4) Gene replication by PCR
               • 5) Genes are sequenced and those with errors are refected
                 • 6) Correct genes are transferred to host cell
   • Preparing a DNA fragment for insertion into host cell
     • Promoter and terminator required
       • Promoter =
         • Located in front of gene
         • Specific sequence for binding of transcription factors
       • Terminator =
         • Location where DNA polymerase disengages from DNA
     • Place a promoter in front of the gene and a terminator behind the gene

2. In vivo cloning - Use of vectors

   • In vivo = performed or taking placed in a living organism
   • In vitro = in glass
   • A vector is something which carries DNA into a cell
   • How to add DNA fragment into a Vector:
     • 1) cut vector with sticky ends using restriction endonucleases
       • 2) Cut the insert with sticky ends with restriction endonucleases
         • 3) mix together
           • 4) single stranded DNA overhands complementary base pairs
             • 5) add DNA ligase to connect sugar-phosphate backbone on both strands
   • Advantages:
     • Once DNA is in vector, it is easy to transform
     • only gene complementary to sticky ends cloned
     • Clone specific genes
     • bacteria make multiple copes of gene

3. Introduction of DNA into host cells

   • By inserting the DNA into the host cell, every time it replicates it will have the DNA
   • Process of insertion is called transformation
   • Transformation can only occur when:
     • Host bacterial cell is mixed with calcium ions to make them more permeable
     • Chill in ice, then warmed to 42 degrees C
   • After transformation, not all bacteria take up the DNA because:
     • Some plasmids may not contain the insert at all
     • Sometimes DNA fragments join together to form plasmids

4. Screening for genetically modified cells

   • 1) Antibiotic resistance Marker Genes
     • Cut plasmid with same RE used to produce gene fragment
       • Add fragment and DNA ligase to cut plasmid
         • Everywhere the ampicillin resistance gene goes, the gene of interest goes too, as they are joined together
           • Recombinant plasmid
     • When diluted and plated onto agar plates containing ampicillin, the two plates marked will die
       • This leaves only the plate with the recombinant plasmid in, so these can be grown and used
   • 2) Fluorescence Marker Genes
     • Screening for loss of fluorescence:
       • 1) Original plasmid contains both antibiotic resistance and fluorescence protein gene
         • 2) Gene of interest is inserted into centre of fluorescence gene
           • 3) diluted bacteria spread onto agar plate containing Ampicillin
             • 4) bacteria that do not glow green are selected
     • Screening for gain of fluorescence:
       • 1) Original plasmid contains only antibiotic resistance gene (Ampicillin)
         • 2) the gene of interest together with the fluorescence gene is ligated into a plasmid
           • 3) Diluted bacteria are spread onto agar plate containing Ampicillin
             • 4) Bacteria that glow green are selected
   • 3) Enzyme Marker Genes
     • Screening for loss of enzyme:
       • 1) original plasmid contains both antibiotic resistance (Ampicillin) and LacZ enzyme gene
         • 2) The gene of interest is inserted into the centre of LacZ gene
           • 3) Diluted bacteria are spread onto an agar plate containing Ampcillin and substrate
             • 4) Bacteria that do not hydrolyse the substrate are selected
     • Screening for gain of enzyme:
       • 1) original plasmid contains only an antibiotic resistance gene for Ampicillin
         • 2) gene of interest is inserted together with LacZ
           • 3) Diluted bacteria are spread onto agar plate containing Ampicillin and substrate
             • 4) Bacteria that hydrolyse the substrate are selected

5. RE = restriction endonuclease

6. In Vitro gene cloning PCR

   • To replicate DNA in vitro you need:
     • DNA fragment
     • pair of primers
     • Nucleotides
     • DNA polymerase
     • Thermocycler
   • How PCR works:
     • 1) Strand separation - DNA heated to 95 degrees to break H bonds
       • 2) Primer binding - cooled 55 degrees to allow primer to anneal
         • 3) Strand synthesis - heat it to 72 degrees DNA polymerase, two strands of DNA complementary to original DNA
   • Advantages:
     • make a lot of DNA
     • Fast
     • can be used for forensic analysis
     • No need for culture cells
   • Disadvantages:
     • Risk of contamination
     • Risk of mutation

7. Relating recombinant DNA technology to gene therapy

   • Somatic cells = non-repoductive
     • Germ cells = reproductive cells
   • Somatic cell therapy targets tissue
     • Germ cell therapy occurs in fertilised eggs
   • Cystic Fibrosis
     • Caused by deletion mutation
     • Chlorine ions are kept inside the cell so osmosis cannot occur as well, less water will leave the cell
       • Therefore, mucus builds up so infections cannot be removed
     • Gene therapy can replace the defective gene with healthy gene
       • Gene supplementation, several healthy genes are placed alongside the defective gene and the dominant healthy gene will be expressed
     • Delivering healthy genes to sufferers:
       • 1) Use of an adenovirus
         • a) Virus rendered harmless by removal of some genes
           • b) Virus grown in an epithelial cell culture together with plasmids and healthy gene
             • c) Plasmids enter the adenovirus DNA
               • d) Virus are extracted from the cell culture and inserted into the noses of patients
                 • e) The virus injects its DNA into the epithelial cells of the lungs
         • Effects are short lived, can cause infections, patients can develop immunity to adenovirus, genes not always expressed
         • Viruses can target specific cells and can adept their own methods of inserting into DNA
         • With more treatments, there is a better immune response, so it is less likely to be taken up
           • Antibodies may attack the virus
       • 2) Use of a liposome
         • Liposome aerosol may not be fine enough to pass through bronchioles
         • CFTR gene
           • Gene in vector
             • Multiple copies are made of vector and insert
               • Cloned copies of the vector placed into liposome
                 • Liposomes passed into lungs via an aerosol
                 • This makes the plasmid lipid soluble
         • Very few of the genes are expressed

8. DNA probes

   • This is a:
     • Short sequence of DNA
     • Complementary nucleotide sequence of gene of interest
     • Radioactive or fluorescence label added
   • Radioactive probe causes film to have a dark shadow where the radioactive label has been
   • Process of locating a specific allele of a gene:
     • Sequence of nucleotides on mutated gene is determined by DNA sequencing
       • Fragment of DNA with complementary bases to the mutant allele of the gene is produced
         • DNA probe is formed by fluorescently labelling the DNA fragment
           • PCR techniques are used to produce multiple copes of DNA probe
             • Probe is added to single stranded DNA fragments from the person being screened
               • If the donor has a mutated gene, some donor DNA fragments will be complementary to the probe, so the probe will bind
                 • These DNA fragments will now be labelled with the probe and can be distinguished
                   • If complementary fragments are present, the DNA probe will be taken up and the dye will fluoresce.
   • The more binding with the probe and the DNA (so hence more fluorescence) shows that more of that gene is present
     • e.g.
       • Where the probe is complementary to cystic fibrosis (allele = a)
       • Aa
         • aa
           • AA
       • Aa = carrier aa = disease AA = healthy
   • Screening for tumour suppressor gene is important because:
     • Mutations of both alleles will result in inactive tumour suppressor
     • Screening may identify hereozygous individuals for gene, so higher chance
   • This allows for personalised medicines:
     • Medicinal treatments can be based on genotype
     • Some patients genotype means drugs may be more/less effective
     • Tailer drug selection and dosage to each individual
   • Genetic counselling is the process by which patients at risk of an inherited disorder are advised of the consequences and probability of having children

9. DNA fingerprinting

   • Some parts of non-coding DNA contain short sequences of repeated DNA
   • The technique:
     • Extraction - DNA extracted from sample
       • Digestion - restriction endonucleases cut the DNA into fragments
         • Separation - Fragments separated with electrophoresis
           • Separation - DNA fragments transferred from gel to nylon membrane
             • Hybridisation - DNA probes added to label fragments
               • Development - Membrane with radioactive labelled DNA fragments placed onto film
                 • Development - Reveals dark bands where DNA probes attached
   • Red blood cells cannot be used for DNA fingerprinting because they have no nucleus
   • DNA must be washed with dilute acid after the fragments are on the nylon membrane to make it single stranded
     • This is because it must be single stranded to allow the probe to bind

10. Describing how genetic fingerprinting works in an exam:

    • 1) DNA is cut
      • 2) Using restriction enzyme
        • 3) Electrophoresis
          • 4) Separates according to size
            • 5) DNA made single stranded
              • 6) Transfer to nylon membrane
                • 7) apply probe
                  • 8) radioactivity detected on film
                    • 9) Pattern is unique to every individual

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