2. California Science Standards #5c,d,e DNA Fingerprinting “Elementary, my dear Watson.” -Sherlock Holmes

3. What is it?  DNA fingerprint = A pattern of bands; each band is a fragment of DNA of a particular size. Each person has a unique DNA fingerprint (except identical twins). This is similar to each person’s actual fingerprint being different. DNA fingerprinting has many applications.

4. What does it look like? DNA samples taken from 2 suspects DNA collected as evidence from crime scene

5. What’s it good for?  1. Determine whether two individuals are related (“Who’s Yer Daddy?”) Compare banding patterns from two or more individuals May be used in paternity cases, or to determine if someone is a long lost relative, etc. The military compares DNA of deceased soldiers to a family member or to a database to confirm identity

6. What’s it good for?  2. Help solve a crime Compare samples of blood or tissue found at a crime scene with a suspect’s blood sample May be used to identify a victim Has also been used to exonerate (clear) individuals wrongly convicted, many whom were serving time on death row.

8. What’s it good for?  3. Determine how closely two species are related (evolution research) Compare banding patterns from members of two or more different species  4. Monitor populations: Tracking grizzly bears in Glacier Nat’l Park. Determining if farm-raised salmon mate with native salmon Determine amount of genetic diversity that exists in a small population, such as an endangered species.

9. How is DNA fingerprinting done?  Just four “easy” steps: 1.Isolate DNA from a cell and cut DNA into many fragments by restriction enzymes (“digestion”) 2.Separate DNA fragments by gel electrophoresis 3.Bind Radioactive probes to selected fragments * 4.Photograph radioactive probes, producing the actual “DNA fingerprint” * * Note: our lab will involve a slightly simpler procedure. The number of fragments is small, so no probes are needed. Also, the “DNA” can be visualized directly without radioactive probes.

10. 1. “Digestion” a)Extract DNA from blood or other tissue (ex: cheek cells, hair follicles, skin, etc.) Remember, all of these cells, taken from the same person have the exact same DNA. b)Cut it into fragments using restriction enzymes.  These enzymes were first discovered in bacteria. Again, we see bacteria are beneficial!  Each restriction enzyme recognizes a specific nucleotide sequence.  Result: the number of fragments and the lengths of the fragments vary from person to person. Recall that each person’s DNA sequence is unique (except identical twins), especially in “noncoding” regions.

11. Example of Digestion The enzyme “EcoR1 recognizes the DNA sequence on the left. In 3 different people, this sequence will occur a different number of times and/or at different locations along a stretch of DNA. After digestion, Bob’s DNA would be in 2 fragments, Larry’s would be in 3 fragments, and Mary’s DNA would not produce fragments.

12. 2. Gel Electrophoresis From the Latin electrocus (“electricity”) and the Greek phoresis (“to carry”) This procedure will use electricity to separate the DNA fragments based on their size. DNA is negatively charged which will cause it to be attracted to (move toward) the positive electrode.

13. 2. Electrophoresis (continued) a)Make a gel (kinda like Jello) b)Make wells in gel c)Inject sample containing DNA fragments into wells d)Run electric current through the gel e)(-) DNA fragments’s move to (+) end of gel but at different speeds: Smaller DNA fragments migrate faster and further than longer fragments

15. Result of Electrophoresis  Diagram of an Example:

16. Actual DNA Fingerprint Each “lane” (column) represents a different DNA sample.

17. 3. Bind Radioactive Probes  Goal of this step: make some bands visible (we only care about certain fragments) a)Split the migrated DNA fragments into single chains b)Blot onto filter paper c)Add complementary segments of DNA to paper These probes are “hot” -- radioactively labeled The probes bind to their complements on paper (they will only bind to some of the fragments, thus not all fragments will be visualized.)

18. Probes http://www.accessexcellence.org/AE/AEPC/NIH/images/probe.gif

19. 4. Photograph  Take a picture: a)Expose photographic film to blot Since radioactive substances show up on film, the radioactive probes show up in the picture when the film is developed b)Develop film The dark spots are the locations of each of the “tagged” DNA fragments (fragments to which the probes attached) Or, if the background is black, the fragments appear as white lines (kinda like an x-ray). c)Analyze DNA fingerprint By comparing the dark bands, we are actually comparing the DNA of the different samples

20. Well, Watson? Who Dunnit?

21. A Tool for C.S.I.  PCR  Polymerase Chain Reaction Often, there are only tiny amounts of DNA at a crime scene. But it’s still enough: PCR “amplifies” the DNA such that enough copies are produced to allow for analysis.