1. Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer?

2. Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer?

3. Bioinformatics/PCR Lab Important Concepts: genetic tests (molecular) genetic marker sequence (DNA) RFLP ( restriction fragment length polymorphism ) genetics and disease greater risk vs. known association population studies

4. Bioinformatics/PCR Lab Sampling of Genetic Tests Available -Alzheimer ’ s Disease* -Lou Gehrig ’ s Disease (amyotrophic lateral sclerosis) -breast/ovarian/colon cancer* -cystic fibrosis -fragile X syndrome -Huntington ’ s Disease -sickle cell anemia -Tay-Sachs Disease *susceptibility test only

5. Bioinformatics/PCR Lab Linking Genetics to Multifactorial Disease (greater risk; involves population studies) -cancer -other current avenues of research peripheral arterial disease type I diabetes schizophrenia/bipolar disorder calcium oxalate stone disease Chron ’ s disease ulcerative colitis

6. Bioinformatics/PCR Lab How does having a certain genetic marker affect chances of getting brain cancer? How does having a specific nucleotide sequence in a specific region of DNA affect chances of getting brain cancer?

7. Bioinformatics/PCR Lab How do you determine what the nucleotide sequence is in a piece of DNA? How do you detect differences in nucleotide sequences in a piece of DNA? How do you determine if these nucleotide differences are related to disease risk?

8. Bioinformatics/PCR Lab Bioinformatics: human genome project ( link in course website ) 1990-2003 identify all human genes sequence human chromosomes make available to all (databases)

9. Bioinformatics/PCR Lab Bioinformatics: human genome project ( link in course website ) comparisons of sequence information within species across species other species genomes sequenced “ They ’ re Sequencing a What? ” Science News, 10/9/04, pp. 234-36.

10. Bioinformatics/PCR Lab Bioinformatics: human genome project ( link in course website ) comparisons of sequence information BLAST search results? specific DNA segment to work with? “ real ” interest in this DNA? why are we using this segment?

11. Bioinformatics/PCR Lab General Lab Procedures: 1) isolate DNA from hair ( “ hair digest ” ) 2) amplify (make many copies of) DNA from hair using PCR 3) cut PCR product with special enzymes restriction enzymes “ restriction digest ”

12. Bioinformatics/PCR Lab General Lab Procedures: 4) visualize cut PCR product on a gel 5) compare patterns (RFLPs) 6) follow people around for decades to see who (which RFLP) gets brain cancer!

13. Bioinformatics/PCR Lab Specific Lab Procedures: 1) isolate DNA from hair ( “ hair digest ” ) hair with root 400  l “ master mix #1 ” (buffer + proteinase K) 1 hour at 65 o C; 10 ’ at 100 o C

14. Bioinformatics/PCR Lab Specific Lab Procedures: 2) amplify (make many copies of) DNA from hair using PCR “ hair digest ” tube containing isolated DNA PCR tube 50  l “ master mix #2 ” transfer 50  l overnight in thermal cycler

15. Bioinformatics/PCR Lab Specific Lab Procedures: 3) restriction digest of PCR product PCR tube containing “ PCR product ” (many copies of desired DNA) 15  l “ master mix #3 ” (buffer + PalI) transfer 5  l 1 hour at 37 o C

16. Bioinformatics/PCR Lab Specific Lab Procedures: 4) visualize cut PCR product on a gel “ undigested PCR product ” “ digested PCR product ” new 1.5ml microfuge tube transfer 15  l DNA loading dye transfer 5  l

17. Bioinformatics/PCR Lab Specific Lab Procedures: 4) visualize cut PCR product on a gel “ undigested PCR product ” “ digested PCR product ”

18. Bioinformatics/PCR Lab Specific Lab Procedures: 5) compare patterns (RFLPs) RFLP #1RFLP #2

19. Bioinformatics/PCR Lab Specific Lab Procedures: 6) follow people around for decades to see who (which RFLP) gets brain cancer! RFLP #1RFLP #2 if people with RFLP #2 have higher incidence of brain cancer, then RFLP #2 becomes a genetic marker for brain cancer …

20. Bioinformatics/PCR Lab Techniques to Know: 1) PCR 2) Restriction Digest 3) Gel Electrophoresis

21. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction method of making many copies (amplifying) of a specific sequence (region) of DNA in vitro (in a test tube) developed by Kary Mullis, 1983 Nobel Prize in Chemistry, 1993

22. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction DNA Replication in vivo (in a cell) Helicase SSBs Primase (RNA primers) DNA polymerases Ligase Nucleotides ( dNTPs, rNTPs ) DNA template DNA Replication in vitro (PCR) Not needed Pre-made DNA primers DNA polymerase (Taq) Not needed Nucleotides ( dNTPs ) DNA template Thermal Cycler

23. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction Thermal Cycler: cycles through 3 different temperatures 30 times each temperature = different step of PCR 1)Denaturation (~94 o C; 30 seconds) 2)Annealing (~50 o C; 30 seconds) 3)Primer extension (72 o C; 1 minute)

24. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) dsDNA template separates into two ssDNA cellular function: helicase, SSBs

25. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) 2) Annealing (~50 o C; 30 seconds) pre-made DNA primers H-bond to template define region to be amplified one primer for each strand of template orientation correct with respect to 3 ’ /5 ’ cellular function: primase, primer removal, ligase

26. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction 1)Denaturation (~94 o C; 30 seconds) 2) Annealing (~50 o C; 30 seconds) 3) Primer extension (72 o C; 1 minute) Taq DNA polymerase adds nucleotides to 3 ’ OH starts from primer cellular function: same

27. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction In the olden days … 3 waterbaths at each temperature Fresh DNA polymerase added each cycle Automation Thermal cycler controls temperature Taq DNA polymerase remains stable Taq = Thermus aquaticus

28. Bioinformatics/PCR Lab PCR: Polymerase Chain Reaction Animation: www.dnalc.org/shockwave/pcranwhole.html

29. Bioinformatics/PCR Lab Restriction Digest Restriction enzymes Restriction digest = fragments of certain length due to specific sequences

30. Bioinformatics/PCR Lab Restriction Digest Recognizes “ the ” and cuts between h - e themonkeyandthedonkeyaretherenow (1) th emonkeyandth edonkeyareth erenow (4) themonkeyandthedonkeyarethreenow (1) th emonkeyandth edonkeyarethreenow (3)

31. Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules based on their physical/chemical properties through a matrix by applying an electric current

32. Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties through a matrix by applying an electric current

33. Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties size, charge through a matrix by applying an electric current

34. Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids, proteins based on their physical/chemical properties size, charge through a matrix agarose, polyacrylamide by applying an electric current

35. Bioinformatics/PCR Lab Gel Electrophoresis Separation of molecules nucleic acids (DNA) based on their physical/chemical properties size (length in base pairs - bp) through a matrix agarose by applying an electric current

36. Bioinformatics/PCR Lab Gel Electrophoresis Before - samples in sample wells After - samples moved through gel ++

37. Bioinformatics/PCR Lab Gel Electrophoresis -why does DNA migrate to + end? +

38. Bioinformatics/PCR Lab Gel Electrophoresis -size vs. location of DNA bands on gel? +

39. Bioinformatics/PCR Lab Gel Electrophoresis -how do you know size of DNA fragments? + Standards of known DNA size (bp) are loaded onto the gel “ DNA ladder ” 1000 800 600 400 200