1. Genetic Testing Family Secrets Copyright © 2009, University of Rochester

2. Genetic disease and testing The patient’s journey… Types of genetic tests Techniques used in genetic testing Implications of genetic testing The future…

3. Inherited disease

4. x23

5. Genetic disease

6. Symptoms

7. ?

8. Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life. Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes) ??? ? Genetic Testing

9. Predictive testing Tells a person if she carries a mutation that will cause, or put her at higher risk for, a disease later in life. Newborn screening Detects common disorders in newborns, where immediate treatment can prevent dangerous symptoms Carrier testing Tells a person whether or not he carries a mutation that could be passed on to his offspring. One can be a carrier, but not be at risk for a disease (as in recessive genes) ??? ? Genetic Testing

10. ? Physician Genetic Counselor Test family members with disease symptoms? Test patient?

11. ? Treatment Prevention Reproduction Physician Genetic Counselor

12. Genetic Testing: Molecular Techniques

13. DNA RNA Protein Function

14. DNA RNA Protein Function Genetic testing

15. DNA Protein Function Levels of Genetic Testing normalmutated Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions atcgatcgatcgatcgaAcgatcg Analysis of sequence – for small changes; mutations in the sequence, small deletions or insertions Analysis of protein shape – for any change that may affect the folding of the protein Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product XX

16. DNA Protein Function normalmutated Analysis of whole chromosomes – for large changes; extra chromosome, very large deletions or insertions atcgatcgatcgatcgaAcgatcg Analysis of sequence – for small changes; mutations in the sequence, small deletions or insertions Analysis of protein shape – for any change that may affect the folding of the protein Analysis of protein function – if the functional protein is supposed to make something, then some tests can detect the presence or absence of the product XX Levels of Genetic Testing

17. Examples of Mutations in the DNA Sequence…

18. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc Normal Examples of Mutations in the DNA Sequence…

19. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgataatcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc Single base pair mutation Examples of Mutations in the DNA Sequence… (Sickle cell anemia)

20. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc Deletion Examples of Mutations in the DNA Sequence… (Cystic fibrosis)

21. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatc tactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggcta tattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacgag gcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacacag cgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagct agtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactagagctac tacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcggatgatc tatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatc Deletion Examples of Mutations in the DNA Sequence… (Duchenne muscular dystrophy)

22. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcaaaaaaaaaaaaaaaaaaggtttttttttttcagctagct tacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgat cgacataaaaaaaaaaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaacc atctaggctatattcggatatcgatctagatatcgatctatcggatctatctactagagctactacgatcagggatatc gatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacg atcaggatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacg agcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgt ggggggggacacagcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaa aaaaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatc tatctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctag gctatattcggatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatcta ggctatattcggatatc Insertion Examples of Mutations in the DNA Sequence… (Huntington’s disease)

23. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc ggatatc Multiple mutations Examples of Mutations in the DNA Sequence… (Diabetes, susceptibility to breast cancer)

24. aaaccatctaggctatattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaaaaaaacgtgagctagtg atgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcggatatcgatctatcggatct atctactagagctactacgatcagggactactacgagcatcgactacgaggcttctagaggctatattctaggcta ctacgatcgatctacgtagctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcaa aggtttttttttttcagctagctggggggggggggatcgggtgtgtcgatgtgtgagcaaaatattagcaacccccc ccccattactgatgtcattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacga gcatcgactacgaggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtg gggggggacacagcgatctaatataaatctgatggtcaaaggtttttttttttcagctagcttacgatcgatctacgta gctacgagatcgtgtgtggggggggacacagcgatctaatataaatctgatgatcgatcgacataaaaaaaaaa aaaaacgtgagctagtgatgggtgatgtcagtgtagtcgtagtcgtgtgataaaaaaccatctaggctatattcgg atatcgatctagatggggatctatcggatctatctactagagctactacgatcagggatatcgatctatcggatctat ctactagagctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggct atattcggatatcgatctatcggatctatctactagagctactacgatcagggactactacgagcatcgactacga ggcttctagaggctatattctaggctactacgatcgatctacgtagctacgagatcgtgtgtggggggggacaca gcgatctaatataaacacagcgatctaatataaatctgatgatcgatcgacatttttttttaaaaaaaaaaaaaaacg tgagctagtgatgggtgatgtcagtgtagtcgtagtcgtacgatcagggatatcgatctatcggatctatctactag agctactacgatcagggatatcgatctatcggatctatctactagagctactacgatcaggatctaggctatattcg gatgatctatctactagagctgatctatctactagagctgtcgtagtcgtgtgataaaaaaccatctaggctatattc ggatatc Multiple mutations Examples of Mutations in the DNA Sequence… (Diabetes, susceptibility to breast cancer)

25. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Dot blot –Microarray Gel electrophoresis PCR

26. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Dot blot –Microarray Gel electrophoresis PCR

27. Gel Electrophoresis Separates DNA fragments on the basis of size Large fragments take longer than small fragments to migrate through an agarose gel

29. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Southern blot –Dot blot –Microarray Gel electrophoresis PCR

30. PCR (Polymerase Chain Reaction) aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta

31. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Melting 94°C

32. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Annealing 58°C cgggcat aactagg

33. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Elongation 72°C cgggcat aactaggaatcgaatgtgcccgtacgattcgatgcga gctaagctacgctttgatcctcgggatagcta

34. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Melting 94°C cgggcat aactaggaatcgaatgtgcccgtacgattcgatgcga gctaagctacgctttgatcctcgggatagcta

35. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Annealing 58°C cgggcat aactaggaatcgaatgtgcccgtacgattcgatgcga gctaagctacgctttgatcctcgggatagcta cgggcat aactagg

36. PCR aatcgaatgtgcccgtacgattcgatgcgaaactaggagccctatcgat ttagcttacacgggcatgctaagctacgctttgatcctcgggatagcta Elongation 72°C cgggcat aactaggaatcgaatgtgcccgtacgattcgatgcga gctaagctacgctttgatcctcgggatagcta cgggcat aactagg gcccgtacgattcgatgcga gctaagctacgctttgatcctcgggatagcta aatcgaatgtgcccgtacgattcgatgcga gctaagctacgctttgatcc

37. PCR Makes a lot of copies of a specific portion of DNA

38. PCR test for deletion Electrophoresis

39. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Southern blot –Dot blot –Microarray PCR Gel electrophoresis

40. Sequencing aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg “Known gene sequence:” ******************************************* Patient DNA sequence How do we find out if the patient’s sequence is different?

41. Sequencing ******************************************* ttttttttactactactac* tttttttt ******************************************* tttttttt actac* actactactactag* actactactactacgactact* actactactacta*

42. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta*

43. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta*

44. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta*

45. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta*

46. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta* a

47. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta* ac

48. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta* tac

49. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta* taca

50. Sequencing ******************************************* tttttttta* ******************************************* ttttttttactac* ttttttttact* ttttttttac* ttttttttacta* tacac…c…

51. Sequencing actactactactagtactactactactactactacactactactactagtactactactactactactac ******************************************* aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg “Known gene sequence:”

52. Sequencing actactactactagtactactactactactactacactactactactagtactactactactactactac aaaaaaaatgatgatgatgatcatgatgatgatgatgatgatg aaaaaaaatgatgatgatgatgatgatgatgatgatgatgatg “Known gene sequence:” Mutation

53. Issues with Sequencing Informative Time consuming Expensive

54. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Southern blot –Dot blot –Microarray PCR Gel electrophoresis

55. RFLP Analysis RFLP (Restriction Fragment Length Polymorphism) analysis relies on the use of restriction enzymes These enzymes recognize specific DNA sequences (usually palindromes) and cut them…

56. RFLP Analysis Restriction Fragment Length Polymorphism means that there are polymorphisms (differences) between people in the number of restriction sites (and therefore the length of the cut fragments). This person has 4 fragments after restriction digest. This person has a mutation that eliminates one of the sites that the restriction enzyme cuts at. Therefore, this person has 3 bands, one of them being much larger than the rest. If this mutation was associated with a disease, a restriction digest would show that a carrier of the mutation had 3 fragments instead of 4

57. RFLP Analysis

58. Genomic DNA is very long and contains a lot of restriction enzyme cut sites PCR makes many copies of a small region of DNA, better for RFLP analysis

59. RFLP Analysis

62. Issues with RFLP analysis Can not detect all mutations – the mutation has to coincide with a restriction enzyme cut site

63. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Dot blot –Microarray PCR Gel electrophoresis

64. Probes DNA bases pair with complementary bases

65. Probes Probe A probe is an a short fragment of DNA that is complementary to part of a longer DNA sequence. DNA strands can be separated with high heat. As the temperature is lowered, smaller fragments that have complementary sequences (probes) will base pair faster than the long original strands of DNA

66. Probes The ability of a probe to bind depends on: *Its complementarity to the DNA strand it’s binding to… -Single base pair differences can affect binding depending on… *The stringency of the binding conditions -Temperature -Buffer conditions Low stringency High stringency

67. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Dot blot –Microarray PCR Gel electrophoresis

68. Dot Blot (A Southern blot without the gel…) A patient’s DNA (genomic DNA or PCR products) is denatured and spotted to a membrane…

69. Dot Blot Membrane is washed in a solution that contains radioactively labeled probe Probe binds to complementary sequence

70. Dot Blot Membrane is washed in a solution that contains radioactively labeled probe Excess probe is washed off – only probe bound to DNA on the membrane remains Probe binds to complementary sequence

71. Dot Blot The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed…

72. Dot Blot The membrane is exposed to autoradiography film.. Therefore, wherever there is radioactive probe, the film will be exposed… And a black dot will be developed where the probe was…

73. Dot blot to diagnose cystic fibrosis (CF) Normal Normal probe Mutant probe CF patient (homozygous mutant) CF carrier (heterozygous mutant) DNA from a patient is spotted out twice, one will be used with a probe complementary to the normal sequence, the other will be used with a probe that is complementary to a mutated sequence. Since a heterozygote has one copy of the normal gene and one copy of the abnormal gene, both probes can bind, but only half as much binds, making the dot lighter.

74. Issues with dot blots Can be used to test patient for multiple mutations Single base pair mutations may be hard to detect with dot blots

75. How do we “look” at the DNA sequence? Sequencing RFLP analysis Probes –Dot blot –Microarray PCR Gel electrophoresis

76. Microarray (High throughput dot blots. Allows for testing of many different mutations…) Microarrays start with a “chip” on which is spotted many different probes for different mutations. Each dot represents a different probe. If this were a dot blot, it would be called a “reverse dot blot” because the PROBE is spotted, rather than the DNA.

77. Microarray (High throughput dot blots. Allows for testing of many different mutations…) The chip is treated much like a southern or a dot blot, except it is washed with a patient’s DNA (fragmented with restriction enzymes, denatured and labeled with a flourescent dye), and it automated.

78. Microarray Binds to a probe for a CF mutation Binds to a probe for a colon-cancer susceptibility mutation Binds to a probe for a breast-cancer susceptibility mutation If a patient’s DNA binds to spotted probes, a computer detects this by the measuring the intensity of the flourescence emanating from that spot. A homozygote for a CF mutation will have a more intense spot than a heterozygote for the same mutation

79. Issues with Microarrays Expensive

80. Available Predictive Tests Cystic Fibrosis Tay Sachs Disease Lou Gherig’s Disease (ALS) Huntington’s Disease Catastrophically high cholesterol Rare Cancers Inherited susceptibilty to cancer Breast Cancer Colon Cancer Thyroid Cancer

81. Benefits of Genetic Testing If Negative Relief Fewer health check- ups and tests that go with being in a family that tends to have a higher risk of a particular genetic diseases If Positive Able to make informed decisions May be possible to reduce the risk of coming down with severe symptoms

82. Limitations of Genetic Testing Mutations may not always lead to disease Existing tests only look for the most common mutations, some disease causing mutations will not be detected by conventional tests Small chance of errors in testing procedure Testing is not always matched by treatment

83. Limitations of Genetic Testing Mutations may not always lead to disease Existing tests only look for the most common mutations, some disease causing mutations will not be detected by conventional tests Small chance of errors in testing procedure Testing is not always matched by treatment

84. Should I get tested? What will I do if it’s positive? What will we do if he’s positive? Testing is not always matched by treatment: Confidentiality Health Insurance Employers Family

85. Where do we go now? Try to resolve ethical, legal and social issues –Experts in science, ethics, law. –Policy makers Develop new, cheap, simple, effective and accurate tests –Testing for all Advance research in disease treatment –Gene therapy –Drug treatment