Topic 8 - Gene technology

• What are proto-onco genes? Why is it an issue if they get mutated?

  • Genes which can be turned on to stimulate cell division
  • If these get mutated so there always turned on -> uncontrolled cell division -> growth of tumours -> cancer
• What are tumour-surpressor genes? Why is it an issue if they get mutated?

  • Genes which slow cell division when turned on
  • If mutated so there always turned off then it can lead to uncontrolled cell division -> formation of tumours -> cancer
• Define genome

  All the genetic information (genes + alleles) in an organism
• Define protenome

  The complete number of proteins which can be produced by a cell/organism
• Outline how DNA probes can be used

  1. DNA is extracted from the sample and is amplified using PCR
  2. The amplified DNA the undergoes gel electrophoresis
  3. This is then combined with the DNA probes and is transfered to a nylon mesh
  4. Here the target gene and DNA proves combine (if complementary)
  5. These can then be identified to see which genes are present
• What markers could be attached to a DNA probe? Why are they necessary?

  • Fluorescent - UV light can be shined on it to be able to identify the target gene
  • Radioactive marker
• Why are DNA probes useful?

  • Used to identify specific genes
  • Gene counciling - helps to determine the best treatment plan for a patient
  • Some genes for breast cancer don't respond to some treatments
• How is recombinant DNA technology technology to produce insulin?

  1. DNA is extracted from a healthy,insulin producing pancreas and is the extracted using restriction endonucleases
  2. DNA is amplified using PCR
  3. Amplified DNA the transfered into a vector (bacteria)
  4. Bacteria colonies are grown, insulin producing bacteria identified and grown in fermentation tank under optimum conditions
  5. Bacteria then harvested, and insulin is extracted
• How are insulin producing bacteria colonies identified during recombinant DNA technology?

  • Before the DNA is transfered into the vector a marker is attached to it
  • Antobiotic resistance and florescent marker
  • This means insulin/target gene producing bacteria can be identified
• Why is recombinant DNA technology important?

  • Previously insulin harvested for pancreas from pigs (expensive, limited) only one type of insulin so dosnt work for everyone
  • Now many types of insulin can be produced, cheaper and larger more readily available
• Define genetic engineering

  The transfer of DNA / genes for one organism to another so that it has a desire characteristics
• Why is genetic engineering possible?

  • DNA is universal
  • Transcription and translation mechanisms are similar in all organisms
• Formation of DNA fragments using restriction endonucleases.

  • Enzymes that cut/break H+ bonds at a specific recognition sequence ( have a complementary active site
  • Forms a single stranded end of the DNA with unpaired nucleotides 'sticky ends'
• What is a restriction endonucleases?

  An enzyme which can recognise and cut hydrogen bonds at specific DNA sequences
• Describe how a DNA machine is used to produce a DNA fragment

  1. Sequence is designed or fed into a computer
  2. 1st nucleotide is attached to a physical support (bead)
  3. Nucleotides are added in the correct order and are joined in the correct order to prevent branching
  4. Short sections of DNA are formed which are then broken of the support
• Outline the process of PCR

  1. Mixture is heated to 94-96⁰c to break hydrogen bonds between complementary base pairings to form 2 separate strands
  2. Mixture is then cooled to 55-60⁰c , so primers can then anneal to one end of each single strand of DNA, identifying where Taq DNA polymerase will bind
  3. Temperature raised to 72⁰c so Taq DNA polymerase can bind and catalyse the addition of DNA nucleotides by complementary base pairings to produce 2 double strands of DNA
  4. Whole process is then repeated
• Outline the process of genetic fingerprinting

  1. DNA fragments, which have be amplified using PCR are mixed with a loading buffer and are pipette into the Wells of an agar gel
  2. Gel is then placed into a tank and is covered with an alkaline buffer to maintain a constant pH, then a current is applied
  3. The DNA then migrates through the gel (smallest fragments move furthest)
  4. Gels are then removed from the tank and are photographed under a UV light
  5. The bands are then compared to a ladder to identify target genes
• What are VNTR's?

  • Variable number tandem repeats
  • Repeating non-coding sequences which occur at different parts of the genome
• How is a sample prepared for genetic fingerprinting?

  1. Restriction enzymes are used to make DNA fragments which contain VNTR'S
  2. These are then amplified via PCR
• Uses of genetic fingerprinting?

  • Medical diagnosis- genetic diseases
  • Forensic investigation
  • Determining genetic relationships
• Define genetic therapy:

  The manipulation of the genetics of a human cell
• Outline the process of gene therapy in the treatment of cystic fibrosis:

  1. Liposome/vector containing the functional allele are placed into an aerosol inhaler and sprayed into the nose of a patients
  2. Some liposomes will pass through the plasma membrane of the cells lining respiratory tract
  3. Some liposomes pass through nuclear envelop and insert into host genome
  4. Host ce express CFTR protein
  5. Treatment is then repeated every 2 weeks
• What is Geraldine gene therapy?

  • Altering of the genome of the gamete of zygote. Therefor causing the change in genome of any offspring / future generations.
  • Could disrupt expression of any other genes
• Ethical issues with the genetic modification of animals:

  • +Ives - reduces cost and can increase supply. No different than crossbreading
  • -Ives - reduces variation, could be heath implications
• Ethical issues with the genetic modification of plants:

  • +Ives - pest and disease resistance increases crop production
  • -Ives - plants are harder to contain, harm environment, have negative impact of farming
• Ethical issues with the genetic modification of bacteria:

  • +ives - they swap DNA amongst themselves anyway, can produce lifesaving drugs
  • -ives - danger they could escape from the lab, cause mass disruption, increase Antibiotic resistance
• Totipotent stem cell:

  • a stem cell that can become any other type of cell in the body
  • as there are no genes turned off
• Pluripotent stem cell:

  • a stem cel which can become most cells but not all
• Multipotent stem cells:

  • stem cells which can form only certain cells
  • E.g. bone marrow (turn into RBC or WNC or platelets)
• Unipotent stem cells:

  • can only form one type of cell
  • E.g. Heart cells -> heart cell
• In adults, where are stem cells found? And what can they be used for?

  • Bone marrow
  • Can replace faulty bone marrow to help treat leukaemia, sickle-cell anaemia
• How are induced pluripotent stem cells created?

  • Created in a lab
  • By ‘switching on‘ genes that are normally expressed in pluripotent cell
• What are the uses of induced pluripotent stem cells?

  • Allow stem cells to be created without ethical issues
  • In the future could be used to create tissues/organs from the patients own cells without the risk of rejection
• Why are there regulatory authorities for human embryo research?

  To help regulate research and the use of embryos
• What is a cardiomyocite?

  • Muscle within the heart
• What are transcription factors? And what do they do?

  • Proteins which control the rate of transcription
  • Control gene expression
• What is an activator (1) and repressor (2) for gene expression?

  1. Stimulates transcription
  2. Slows/stops transcription
• Describe how oestrogen can affect transcription of a gene?

  • Oestrogen binds to oestrogen receptors forming an oestroge-oestrogen complex
  • Moves into the nucleus and binds to HRE in promoter
  • Activating or repressing transcription
• Why aren’t all cells effected by oestrogen?

  not all cells have oestrogen receptors
• What is siRNA? And how does it work?

  • small interfering RNA
  • It associates to proteins and binds to target mRNA
  • Cuts it into small fragments which can no longer be translated