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Mitochondrial DNA

Mitochondrial DNA. … and its’ use in Forensics. Biology Recap. What is the Mitochondria?. The Mitochondria are often considered the Cell Power house They generate much of the Eukaryotic Cell’s ATP --- (remember ATP is the cell’s energy molecule). Mitochondria are responsible for….

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Mitochondrial DNA

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  1. Mitochondrial DNA • … and its’ use in Forensics

  2. Biology Recap • What is the Mitochondria?

  3. The Mitochondria are often considered the Cell Power house • They generate much of the Eukaryotic Cell’s ATP --- (remember ATP is the cell’s energy molecule)

  4. Mitochondria are responsible for… • Cell Energy Production • Cell Growth • Cell Signaling ( sending cell messages) • Cell Death or Apoptosis

  5. Some Human cells have 1 mitochondria • Others have MANY • Can you think of a human cell that might have hundreds of Mitochondria?

  6. A Muscle Cell! This is a close up of Cardiac Muscle fibers There are hundreds of Fibers in a single cell Each M stands for Mitochondria

  7. Endosymbiotic Theory • The theory that mitochondria and chloroplasts at some point in time were separate organisms from a human cell. • They were engulfed by a cell and became a symbiont

  8. Biologists believe this is how mitochondria and chloroplasts came to be

  9. What is the evidence for this theory? • Well mitochondria and chloroplasts both are separately bound organelles. • More Importantly both chloroplasts and mitochondria have their own DNA

  10. Mitochondria resemble certain kinds of bacteria • They even have a circular DNA molecule or plasmid

  11. Mitochondrial DNA Molecule is 15,000 – 17,000 base pairs long

  12. In mammals all mitDNA consists of the same 37 genes • 13 genes for proteins • 22 genes for transfer DNA (tDNA) • 1 for the small subunit of the ribosome • 1 for the Large subunit of the ribosome

  13. In humans all the Mitochondrial DNA comes from 1 parent • The Mother

  14. Why is this? • The egg cell has between 100,000 and 1,000,000 copies of mitDNA • The sperm cell only has 10 • (when the sperm cell fertilizes the egg, the 10 copies of mitDNA from the sperm are left behind, erasing the mitDNA from his side of the family)

  15. Mitochondrial DNA allows scientists to do a maternal lineage • …or trace a person’s ancestors based on mitDNA • In order to run a paternal lineage scientists would have to trace the Y chromosome… this is a much larger task--- the Y Chromosome is 58 million base pairs long!

  16. Who were the Romanovs? • What happened to the family and their remains? • How did they discover the bodies? • How did they prove the remains in the bog belonged to the Romanovs? • Which Romanovs matched the MitDNA from Prince Philip? Which were missing? • Who was Anna Anderson? • Was Anna Anderson princess Anastasia?

  17. Southern Blot • This is the combination of Gel Electrophoresis and a specific way of probing for a specific DNA sequence • So you separated your sample by size, How can you find Gene X?

  18. Method Part 1 • Perform a RFLP and run your samples on a Gel Electrophoresis to separate your sample by size

  19. Part 2: Blot your sample onto a nitrocellulose membrane • Once your DNA samples are on the membrane, denature it • You can do this by adding a base such as NaOH

  20. Your double stranded DNA will become Single stranded DNA

  21. Step 3: Once this is done you can add your detecting probe to bind with your DNA • A Probe is a specific sequence of DNA

  22. For Example • ATTACGATCCCCATCCACC • If we add a probe that has the sequence AGGGG….where will this bind?

  23. If we add a flourescent marker to our AGGGG tag, then wherever the marker binds there will be a glowing band… like this

  24. Step 4: Wash away excess Probe

  25. Step 5: Detection • Usually this is a visualization of your specific DNA sequence • Audioradiography ( if you used a radio active probe) • Or Luminence if you used a flourescent probe

  26. The main purpose of the Southern Blot is to take a bunch of unknown DNA sequences, and find a specific gene or sequence

  27. Left is Gel Electrophoresis, Right is a Southern Blot…a specific DNA sequence was found

  28. Southern Blot would be used In cases where a suspect is known to have a known genetic defect, such as Cystic Fibrosis Any specific DNA sequence that would provide evidence

  29. The Polymerase Chain Reaction (PCR) Or DNA photo copying

  30. Why would anyone want to make more copies of DNA?

  31. PCR allows us to take a very tiny amount of DNA and multiply this into millions of copies • It lets us decode tiny, trace amounts of DNA – Like in the Amanda Knox case.

  32. Once we have billions of copies, we can sequence the DNA strand and determine who that DNA came from

  33. PCR has allowed us to map segments of the Human Genome that code for rare diseases… • this allows us to do genetic testing – on infants and on you! • this also allows you to be able to find out if you have the gene for Alzheimer's Disease, or Parkinson’s Disease or Cystic Fibrosis or many other genetic diseases

  34. PCR is also a test used to determine if you have the HIV virus in your blood cells.

  35. Kary Mullis • The American Molecular Biologist who invented PCR • 1985 • Won the Nobel Prize in Chemistry in 1993

  36. How does PCR work? • 4 Steps • Denaturing • Annealing • Extension • Exponential Extension

  37. Thermus aquaticus - a very tiny bacteria!! • Thermus aquaticus lives in the nice warm hot springs and thermal vents in Southwestern USA • These hot springs are between 75 and 80 degrees C • Taq has enzymes that function well at these high temperatures • Looks like this!!--->

  38. Taq Polymerase • Poly = many • Ase = to make • This enzyme copies DNA

  39. PCR Step 1: Denature your DNA sample • Heat your DNA sample up to about 98 degrees C, almost boiling for 20-30 seconds • This will make your two DNA strands come apart • How else can you denature DNA?

  40. Double stranded molecule pulls apart at high temperatures

  41. Step 2 Annealing of Primers • “Anneal” means “to bind” • Primers: regions of DNA that are the message to begin DNA replication • Add these DNA segments to our Denatured DNA and let them bind. • *This step also only takes 20-30 seconds*

  42. Step 3: Elongation • Add the Taq polymerase • Add Extra nucleotides (A,T,C,and G) • Set heat for 75-80 degrees C • Time it takes to elongate depends - usually 5-15 minutes • DNA polymerase can elongate DNA at a rate of 1,000 bases/minute!!!

  43. Step 4: Repeat!!! • We repeat these steps, 30-40 times to EXPONENTIALLY CLONE our DNA sample!!!

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