Chap 19: Genetic technology

• Recombinant DNA: genetically altered DNA with an introduced nucleotide.

  Transgenic: organism contains nucleotide sequences from different species.

  Gene: a length of DNA that codes for a protein
• Outline 3 sources from which genes can be obtained for genetic engineering

  1. Genes cut from (donor) DNA
  2. mRNA from (donor) organism
  3. Gene synthesised chemically from nucleotides
• Outline the principles of genetic engineering.

  1. Add DNA to give new characteristic
  2. Restriction enzyme/endonuclease to obtain gene/allele or to cut plasmid
  3. Combine gene with plasmid
  4. Introduce recombinant plasmid to bacterium
  5. Clone/multiply recombinant organism
• Restriction endonuclease

  • Cut DNA
  • Produced by bacteria / to defend against viruses
• DNA polymerase

  • Convert single strand cDNA to double strand DNA
  • Replicates DNA
• DNA ligase

  • Joins DNA forming sugar phosphate backbone (phosphodiester bonds)
  • During DNA replication
  • Joins Okazaki fragments
• Reverse transcriptase

  • Forms cDNA from mRNA
  • Produced by retroviruses
• Taq polymerase

  • Replicates DNA at high temperatures
• Describe how PCR is used to clone and amplify DNA

  1. DNA denatured
  2. By heating to 95 degree celsius
  3. Add primary DNA
  4. Complementary base pairing with sample DNA
  5. At 55-65 degree celsius
  6. Annealing
  7. DNA polymerase builds new strands
  8. By adding free nucleotides
  9. At 70-75 degree celsius
  10. Taq polymerase thermostable
  11. Does not need replacing
  12. New strand denatured and process repeated
  13. Process is automated
• Describe and explain the properties of plasmids that allow them to be used in gene cloning

  1. Double stranded DNA
  2. Have restriction site/target sequence for restriction enzyme
  3. Allow gene to be inserted
  4. Small → plasmid can enter host cell/bacterium
  5. Circular → stable
  6. Contain marker gene or genes for antibiotic resistance → used to identify recombinant cells
  7. replicate independently/fast → get many copies of cloned genes
• Why plasmids can be used as vectors in gene cloning

  1. Small → can be inserted into cells
  2. Replicate independently/fast → high copy number
  3. Has restriction sites/can be cut by different restriction enzymes → new gene can be added
  4. Have multiple cloning site → can be cut by different restriction enzymes
  5. Have marker genes → recombinants can be recognised
  6. Circular → stable
• Similarities between genetic engineering using a plasmid and gene editing.

  • Both can produce transgenic organism
  • Both modify the characteristics of an organism - produce different protein
  • Both uses enzyme (restriction endonuclease) to cut DNA
• Genetic engineering using a plasmid:

  • Does not change base pairs in gene
  • Uses DNA ligase
  • Success can be evaluated by using gene markers
  • Requires DNA donor and recipient
  • Does not inactivate a desired selected gene
  • Cannot use RNA to precisely locate the target gene
• Gene editing:

  • Changes base pairs in gene (insertion, deletion, replacement of DNA): A-T, C-G
  • Uses CRISPR system
  • Does not use gene markers
  • Does not require DNA donor and a recipient
  • Can inactivate a desired selected gene
  • Can use RNA to precisely locate the target gene
• Explain why a promoter also needs to be transferred into the mammalian cells.

  1. Required for gene expression/to start transcription
  2. So transcription factor can bind to promoter
  3. So RNA polymerase can bind to promoter
• How is gel electrophoresis used to separate DNA fragments of different lengths.

  1. Different charge of molecules: Negative DNA moves to positive electrode
  2. Different sizes of molecules: Smaller/lighter fragments move faster/longer distance
  3. Type of gel: different pore size
• How are microarrays used in analysis of genome

  1. Obtain single-stranded DNA
  2. Label DNA with fluorescent dye
  3. Probes on chip/microarray
  4. Each probe is unique to a particular gene
  5. DNA binds to probes → hybridization
  6. Wash off excess DNA after hybridisation
  7. View under UV light/laser scanner/high resolution digital camera
  8. Fluorescence indicates presence of gene
• Challenges of using a virus for gene therapy.

  1. Retrovirus can insert viral DNA randomly into DNA
  2. May cause cancer/side effects/allergic response
  3. Inserted allele/DNA may be inactivated
  4. Virus may not enter target cells
  5. Ineffective immune response against virus
• Describe the social and ethical considerations for DNA analysis

  • DNA analysis not available for everyone
  • Lifestyle change → e.g. lose weight/exercise/stop smoking
  • Early treatment
  • Allows people to plan → e.g. take our medical insurance/retire early
  • Decide whether to have children
  • Results may cause anxiety/stress/depression (if positive) OR reduces worry (if negative)
  • Results may affect ability to get insurance
  • Predictions may not be accurate
• Ethical considerations of using a retrovirus for gene therapy

  • May attach/deliver the gene to the wrong site
  • May result in interference with other healthy gene
  • Results in diseases (e.g. cancer)
• Outline the principles of gel electrophoresis.

  • DNA cut by restriction endonuclease to produce DNA fragments
  • DNA fragments replicated by polymerase chain reaction (PCR) - so DNA fragments are visible on gel
  • Fragments are loaded at the negative end of the well
  • DNA is negatively charged so DNA will be attracted towards anode
  • Due to electrical field/when current is applied
  • Shorter fragments will move faster and travel further
  • Ref. buffer solution added to maintain pH levels
• Marker genes

  • Green fluorescent protein (GFP)
  • Antibiotic resistant gene
• Bioinformatics

  • biological data is collected, organised, manipulated, analysed and stored
• Benefits of bioinformatics

  • Make comparisonsinformation with genomes
  • Provide ifnormation about nucleotide sequences of genes, genomes, amino acid sequences of proteins and protein structure
• Advantages of genetic engineering

  • More cost efficient to produce in large volume
  • Faster to produce
  • Reliable supply available
  • Solve issues of morals, religious and ethical considerations
• Adenosine deaminase

  • adenosine deaminase deficiency
  • cause of severe combined immunodeficiency (SCID)
  • decrease the risk of transmitting infections
  • increase reliable production of enzymes
  • faster to prodce many proteins
• Breast cancer (BRCA1 and BRCA2)

  • BRCA1 and BRCA2 regulate cell growth
  • Person may decide to take preventative measures - e.g. breast removal
  • Person can participate in research and clinical trials - screening at a young age
• Explain why gene editing is more suitable as a potential cure for Huntington's disease

  • Huntington's disease is caused by dominant allele
  • Repeated triplet if CAG
  • Adding a 'normal' recessive allele would not work
  • Gene editing can delete/remove/inactivate HTT allele
  • Ref. surplus of CAG repeats