1. Nuclear DNA and Mitochondrial DNA

2. Purpose
Isolate, amplify, and sequence a piece of DNA from the mitochondria of your own cheek cells.

3. Nuclear DNA Present in almost every cell
Combination from both parents; 23 chromosomes from each parent

4. Mitochondrial DNA
Each cell contains thousands of mt, each containing copies of its DNA
Mt DNA is in larger quantities in a cell
Nuclear DNA is larger in size

5. Mt DNA is 16,569 bases in length and consists of 2 different regions
Coding Region
Produces 13 proteins, 22tRNAs, 2rRNAs needed for cell respiration
This region has very little variability
So everyone’s DNA in this region will be nearly the same sequence of TGCAs

6. Coding Region DNA RNA Protein Trait

7. 610 bp with a lot of variability
Control Region
This region is highly variable within the human population
Consists of 2 subregions
HV1 = 342 bp HV2 = 268
610 bp with a lot of variability

8. Mt DNA is inherited from mom
Every sibling will get their mt DNA from their mother
Why?

9. Sperm contains 23 chromosomes with very little cytoplasm
Why Mom?
Egg contains 23 chromosomes and cell cytoplasm which contains thousands of maternal mt
Sperm contains 23 chromosomes with very little cytoplasm

10. Zygote = Fertilized Egg
When egg and sperm join only female mt survive and are passed onto to new baby.

11. Maternal Inheritance Pattern with Mt DNA

12. Mutations occur in the control region of mt DNA at a regular rate and are passed onto children by the mom.

13. How do we use this information?
We can compare DNA from the controlling region to other living humans
See how related to you are to each other
Compare to prehistoric remains of human fossils
Identify where you DNA originated
Identify ancestral relationships between modern populations
Compare your highly variable regions to other species

14. Mitochondrial Eve
Oldest women who would have donated her mtDNA to every ancestor in the world
Comparisons can be made by how many variations exist between her DNA and your DNA.

15. Isolate DNA from cheek cells Complete a PCR reaction
How is mtDNA isolated?
Isolate DNA from cheek cells
Complete a PCR reaction
Produce millions of extra copies of HV1 on the control region of mtDNA
Send amplified DNA away to be sequenced (Identify the exact sequence of TGCAs in HV1 in your mtDNA)
Compare your sequence ot classmates and database of prehistoric DNA

16. Procedure
Pour 10 ml of sports drink into mouth and vigorously swish for 1 minute.
Expel saline solution into a paper cup.
Swirl to mix cells in the cup and transfer 1 ml (1000 µl) of the liquid to 1.5 ml tube.
Place your sample tube, in a microcentrifuge, and spin for 1.5 minutes.
Carefully pour off supernatant into paper cup or sink. Be careful not to disturb the cell pellet at the bottom of the test tube. A small amount of saline will remain in the tube.

17. Resuspend cells in remaining saline by pipetting in and out.
Withdraw 30 µl of cell suspension, and add to tube containing 100 µl of Chelex. Vortex to mix.
Place tube in a thermal cycler for 10 minutes at 99°C.
After boiling, vortex tube. Place in a microcentrifuge and spin for 30 sec.
Transfer 30 µl of supernatant (containing the DNA) to clean 1.5 ml tube. Avoid cell debris and Chelex beads.
Store your sample on ice or in the freezer until ready to begin Step 12.

18. Use a micropipet with a fresh tip to add 22
Use a micropipet with a fresh tip to add 22.5 µl of the appropriate primer/loading buffer mix to a PCR tube containing a Ready-To-Go PCR Bead.
Use fresh tip to add 2.5 µl of human DNA (from Part I) to reaction tube. Pipet up and down to mix the contents of the tube.
Label the cap of your tube with a number, as assigned by your teacher.
Store all samples on ice until ready to amplify according to the following profile: Denaturing time and temperature 30 sec - 94°C Annealing time and temperature 30 sec - 58°C Extending time and temperature 30 sec - 72°C Hold at -4°C (optional)

19. Use a micropipet with a fresh tip to add 15µl PCR sample/loading dye mixture into your assigned well of a 1% agarose gel.
Load 5 µl of the pBR322-BstNI size markers into one lane of gel.
Electrophorese at 130 volts for minutes. Adequate separation will have occurred when the cresol red dye front has moved at least 50 mm from the wells.
Gel may be stained with 1 µg/ml ethidium bromide for 10 minutes followed by minutes destaining with water.