1. Personalised medicine is based on an individual’s genome. Analysis of an individual’s genome may lead to personalised medicine through understanding the genetic component of risk of disease.

3. 4 pictures 1 word!

4. Personalised Medicine-The future

5. Personal Genome Sequence Complete sequencing of person’s DNA bases – called personal geonomics Why has this become more and more possibly? Faster and cheaper due to techniques like PCR (next lesson!!) Analysis of an individual’s genome may lead to personalised medicine through understanding the genetic component of risk of disease eg. BRCA 1 and 2 genes for breast cancer means 45 to 65% chance of developing breast cancer by the age of 70!

6. Pharmacogenetics Pharmacogenetics is the study of how people's genetic makeup affects their responses to drugs. If a drug is known to produce side effects in some people, pharmacogenetic testing may help identify at-risk patients.

7. Once DNA sequencing has identified the genes involved in a specific disease and established the structure of the protein expressed, pharmacogenetisists try to synthesise a specific effective drug.

8. The drug produced will –bind to proteins involved or –prevent their synthesis by binding to a specific region of the DNA preventing transcription of abnormal mRNA or –by binding to the abnormal mRNA preventing translation eg interfering RNA (RNAi)

9. Making a diagnosis of disease status or risk of disease onset A cell sample from a patient can be screened for the presence or absence of a particular sequence, eg a mutation in a gene. This information can be used to diagnose a condition or describe the likelihood of a condition developing. For example, women with a family history of cancer can be tested for mutations in the BRCA 1 and BRCA 2 genes. Mutations in these genes can increase the risk of developing breast or ovarian cancer.

10. Personalised medicine Studying an individual’s genome may enable doctors to more accurately diagnose a disease and then prescribe the correct drug, at the correct dosage (level) and at the correct time. It may reduce the possibility of the patient suffering adverse reactions. It may further help to identify an individual’s susceptibility to genetic diseases and enable steps to be taken to avoid or reduce the risk of developing that disease.

11. Now do the pharmacogenetic diamond 9 activity

12. Ethical issues If a person’s genome contains genetic ‘markers’ indicating a high risk - who should have access to this information? Employer? Offspring? Life insurer? Most people believe that laws should be introduced to prevent ‘genetic discrimination’

13. Arrays of DNA probes are used to detect the presence of specific sequences in samples of DNA. The probes are short single stranded fragments of DNA that are complementary to a specific sequence. Fluorescent labelling allows detection.

14. What is the link?

15. Basically a DNA photocopier! A technique for the amplification of DNA in vitro (outside the body – like IVF).

16. Allows selective amplification of any fragment of DNA providing the nucleotide sequences flanking the fragment are known Finds a needle in the haystack and then produces a haystack of needles by selective amplification Kary Mullis: Nobel Prize for Chemistry 1993

17. Polymerase Chain Reaction (PCR) minute quantitiesdrop of blood crime scenecell extinct dinosaur.PCR has made it possible to analyse and identify DNA fragments found in minute quantities in places like a drop of blood at a crime scene or a cell from an extinct dinosaur.

18. So it uses are …. Forensics! Genetic testing

19. The problem! Maisy the research meerkat is pregnant, but who’s the daddy? How can we find out?

20. A real problem for a real scientist Johanna Nielsen, a PhD student at the University of Edinburgh and the Institute of Zoology at the University of Cambridge What did she do?

21. She knew that DNA is the genetic material of living things Therefore the DNA sequence of the foetus would have greater similarity with the father than with a male meerkat who was not the father She had two candidate fathers and decided to compare the DNA from the foetus with that of the two possible fathers She decided to use DNA profiling (fingerprinting) techniques, which involve the use of the polymerase chain reaction, or PCR for short

22. Johanna knows that this section of DNA can be of different sizes between meerkats. She wants to compare the DNA at this site between the three samples. The DNA profiling technique: compares the size and sequence of DNA at a specific site between individuals DNA

23. Firstly, she needed to extract the DNA from each potential father and the foetus. What would she need to do to get a DNA sample?

24. PCR process If you wanted to amplify DNA what things would you need? DNA polymerase

25. By using PCR, Johanna can amplify this section of DNA many times.

26. http://courses.scholar.hw.ac.uk/vle/scholar /session.controller?action=viewContent&b ack=topic&contentGUID=0dbebd90-1472- 67b0-663e-8061550d5f73http://courses.scholar.hw.ac.uk/vle/scholar /session.controller?action=viewContent&b ack=topic&contentGUID=0dbebd90-1472- 67b0-663e-8061550d5f73

27. PCR uses the same mechanism for copying the DNA as our cells use for DNA replication. In pairs, remind yourselves of what happens during DNA replication. What is the main enzyme involved?

28. PCR uses the enzyme DNA polymerase to replicate or ‘amplify’ the DNA strand.

29. Film on PCR procedure http://www.edvotek.com/Resources

30. PCR animations http://www.dnalc.org/view/15924-Making- many-copies-of-DNA.htmlhttp://www.dnalc.org/view/15924-Making- many-copies-of-DNA.html http://www.dnalc.org/resources/3d/19- polymerase-chain-reaction.htmlhttp://www.dnalc.org/resources/3d/19- polymerase-chain-reaction.html http://www.dnalc.org/resources/animations /gelelectrophoresis.htmlhttp://www.dnalc.org/resources/animations /gelelectrophoresis.html

31. Step 1: DNA denatured Primers are used to start the replication process of just the section needed. Primers are short single strands of DNA that bond or ‘anneal’ to the sections of DNA on either side of the section of DNA that is wanted. The temperature is reduced for this step The DNA is heated to 94°C to denature the double helix so amplification can take place. Primers anneal Step 2: Primers anneal The polymerase chain reaction

32. DNA polymerase + Two strands of DNA are produced. Annealed primers Step 2: DNA is copied (Temperature is increased to 72°C)

33. And then start the three steps all over again!...but this time the amplified DNA is also used as a template... To give four...

34. PCR Process Summary – Learn! Repeated cycles of heating and cooling amplify this region of DNA by thermal cycler (~30 times) Heated to 72 o C for DNA polymerase to attach nucleotides Heat tolerant DNA polymerase then replicates the region of DNA. Takes longer for polymerisation of nucleotides Mixture cooled to 50-65 o C Allows primers to anneal/attach to each 3’ end of each strand DNA is heated 94-96 o C Hydrogen bonds between chains break Separate into 2 strands

35. A recap... 30-40 cycles

36. Put the following steps involved in PCR into the correct order. 1.DNA replication occurs using DNA polymerase. 2.Primers introduced and temperature reduced to allow bonding. 3.Temperature increased again to separate strands and the cycle begins again. 4.Temperature increased to 95°C to separate strands. 5.Double-stranded DNA produced, consisting of a template and a new strand of DNA. Draw an annotated diagram to accompany each stage.

37. Share your finished note with a partner and check the steps are in the correct sequence Temperature increased to 95°C to separate strands. Primers introduced and temperature reduced to allow bonding. DNA replication occurs using DNA polymerase. Double-stranded DNA produced, consisting of a template and a new strand of DNA. Temperature increased again to separate strands and the cycle begins again.

38. Maisy’s baby MalcolmMartin After PCR what will be in the sample tubes? Millions of copies of the DNA section/fragment selected by Johanna. Remind yourself why Johanna wanted to amplify this fragment.

39. Johanna needs to find out the size of the DNA fragments inside each sample. She uses a technique called DNA gel electrophoresis, which separates DNA fragments based on their size. In the past this was done in tanks, using agarose gel electrophoresis, but laboratories now use an automated machine that can process many samples quickly using capillary gel electrophoresis.

40. DNA probes DNA probes are pieces of DNA used to detect the presence of specific sequences in DNA. Made of DNA Short Single stranded Complementary to the sequence being searched for

42. http://courses.scholar.hw.ac.uk/vle/scholar /session.controller?action=viewContent&b ack=topic&contentGUID=3c1030b1-0642- a554-5484-24646a7561a2http://courses.scholar.hw.ac.uk/vle/scholar /session.controller?action=viewContent&b ack=topic&contentGUID=3c1030b1-0642- a554-5484-24646a7561a2

43. DNA probes DNA probes are short, single stranded fragment of DNA that is complementary to a specific sequence (called target) DNA Fluorescent labelling allows detection.

44. How do we find where the probes have bound? Before the DNA probe is introduced to the DNA it is fluorescently labelled. This involves attaching a fluorescent dye to the probe. For example, ethidium bromide fluoresces orange when bound to DNA and exposed to UV light.

45. Sequencing DNA A proportion of DNA is chosen and many copies are synthesised. DNA polymerase, primer, the four DNA nucleotides and modified nucleotides are added When a modified nucleotide binds to the DNA strand it halts the process As this is carried out on many copies, eventually all the strands will have stopped at every possible position http://highered.mcgraw- hill.com/sites/0072556781/student_view0/chapter15/ animation_quiz_1.html

46. The results are then processed by a computer, producing data like that shown above. This example shows the results of two different PCR samples. One of the DNA fragments is 121 base pairs in length, the other is 159 base pairs. If the PCR sample contains millions of DNA fragments, why would one sample only produce one data peak?

47. Here are Johanna’s data for three different DNA fragments from each of the meerkats Malcolm Martin Who is the daddy? Martin!

48. Polymerase Chain Reaction (PCR) Many copies of a specific segment of DNA can be produced using a technique called polymerase chain reaction (PCR for short). The copying of this DNA segment is done in vitro – outside the body. DNA is initially heated to break the hydrogen bonds between base pairs, separating the two strands. It is then cooled to allow primers to bind to target DNA. Primers are pieces of single stranded DNA, complementary to a specific DNA sequence that bond or ‘anneal’ to the start and end of the DNA strand to be replicated. DNA polymerase enzymes then add free DNA nucleotides to the primers at the 3’ end of the original DNA strand. This heating and cooling is repeated to create more copies of the DNA. DNA probes are finally used. These are short fragments of single stranded DNA, complementary to a specific sequence, used to detect the presence of specific sequences in the original DNA. Fluorescent labelling of these probes allows visible detection of the strands under UV light.

49. Applications of DNA profiling allow the identification of individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA.

51. Medical and forensic applications In groups discuss how DNA probes along with PCR can be used to: make a diagnosis of disease status or risk of disease onset identify if a particular individual left blood at a crime scene.

52. Crime Scene Forensic scientists make use of PCR to amplify DNA samples from a crime scene. This can then be compared against samples of the victim and suspects

53. Identifying if a particular individual left blood at a crime scene DNA profiling allows the identification of individuals through comparison of regions of the genome with highly variable numbers of repetitive sequences of DNA. So if a tiny sample of blood is left at a crime scene, the DNA it contains can be amplified using PCR. A probe can then be created, which binds to one of these highly variable sequences. If a suspect is apprehended their DNA can be tested with the same probe to look for the sequence in question.

54. Electrophoresis animation http://learn.genetics.utah.edu/content/labs/ gel/http://learn.genetics.utah.edu/content/labs/ gel/

55. Paternity testing Is he the father? Every band in a person’s ‘genetic fingerprint’ must match one in that of their father or mother! Mother ChildFather

56. DNA profiling Complete the alcoholism profiling scenario

57. Applications of DNA profiling By comparing regions of the genome with highly variable numbers of repetitive sequences of DNA, profiling allows individuals to be identified. E.g. blood from a crime scene to a suspect E.g. paternity testing. E.g. a diagnosis of disease or risk of disease onset can be made.