2. DNA Techniques Lab Preparation 13-1

3. Manipulating Genes Genetic Engineering: You can repair genes, insert genes, excise genes or replace genes with gene therapy

4. Manipulating DNA You can identify who was at a crime scene, a John Doe, paternity of children sequence DNA and more with DNA Technology

5. What will we do? We are going to identify who was at the crime scene of a vicious murder of Spongebob Squarepants

6. How do we figure out Who was at the crime scene? Small amounts of trace evidence will be collected In this case, body fluid was found near the body and collected

7. PCR Polymerase chain reaction Sometimes only tiny amounts of evidence are found PCR is like a photocopy machine for DNA, so lots of copies are made

8. What now? The DNA is cut into tiny pieces by chemical scissors known as restriction enzymes (RE) Each enzyme cuts at a specific sequence Each person’s DNA is different, so each RE cuts everybody’s DNA into different numbers and sizes

10. Restriction Enzymes There are lots of restriction enzymes that cut at different sequences

11. Gene Insertion Match the overlapping ends with the appropriate bp and you can insert any gene you want!!!

12. The DNA for this lab has been pre-cut by restriction enzymes

13. WHAT NOW? Because everybody’s DNA is different, determining the sizes of each fragment is as good as a fingerprint!!!

14. How Do We Determine the size of the fragments? Gel Electrophoresis separates the DNA according to size from biggest to smallest with the use of electricity!!!

15. What the heck is gel electrophoresis? DNA is negatively charged Gel electrophoresis takes advantage of that property. We will actually apply an electric current to the DNA

17. AGAROSE A semisolid material that has many purposes in science. It is comparable to a sponge (little holes and big holes)

19. A comb makes wells

21. Fragmented DNA is inserted into the wells

22. Fragment size and agarose Because agar has holes similarly sized fragments will get stuck at about the same places in the agar. This allows us to actually see bands of DNA in the agar

24. Fragment size and agarose If you have big pieces of DNA and little pieces of DNA which do you think would travel the fastest through the agarose? The smaller pieces!!!

26. These are known as DNA fingerprints!!!

28. How do we set up the gel/tray?

33. DYE LAB – TEST RUN FOR GEL ELECTROPHORESIS

34. What did you do well on this lab? How could you improve your skills and how could you reduce the time it took to set up the lab?

36. M=“Marker” (also known as DNA Size Standard – sample of digested DNA with known sizes of fragments

37. Get DNA samples Add 5  l of Loading Dye (LD) to each sample BE SURE TO MIX IN MICROCENTRIFUGE!

38. Put 15  l of sample in each well 23 45MCS 1

39. Interpreting DNA Fingerprint Results

41. Rinse your gel and then view on light box.

45. Ladder/Standard/Marker If we measure how far each set of fragments moved, we can graph the distance (in mm) VS the known fragment sizes (in base pairs) and use this as a kind of molecular ruler

46. Sample

47. Analysis of DNA Fingerprint Data Use your metric ruler to measure distance each band traveled

48. Graph the DNA size standard (AKA “standard marker”) distances VS the given size of the fragments in each band from your table

49. Graph the DNA size standard (AKA “standard marker”) distances

50. Graph the DNA size standard (AKA “standard marker”) distances

51. Who was at the crime scene?

52. Paternity Testing

53. Graph the DNA size standard (AKA “standard marker”) distances