Nuclear DNA and Mitochondrial DNA

Purpose

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

Nuclear DNA

• Present in almost every cell

• Combination from both parents; 23 chromosomes from each parent

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

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

Coding RegionDNA RNA Protein Trait

Control Region

This region is highly variable within the human population

Consists of 2 subregions

HV1 = 342 bp HV2 = 268

Mt DNA is inherited from mom

• Every sibling will get their mt DNA from their mother

• Why?

• Egg contains 23 chromosomes and cell cytoplasm which contains thousands of maternal mt

• Sperm contains 23 chromosomes with very little cytoplasm

Zygote = Fertilized Egg

• When egg and sperm join only female mt survive and are passed onto to new baby.

Maternal Inheritance Pattern with Mt DNA

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

• 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

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.

1. Isolate DNA from cheek cells2. Complete a PCR reaction

– Produce millions of extra copies of HV1 on the control region of mtDNA

3. Send amplified DNA away to be sequenced (Identify the exact sequence of TGCAs in HV1 in your mtDNA)

4. Compare your sequence ot classmates and database of prehistoric DNA

Procedure1. Pour 10 ml of sports drink into mouth and vigorously

swish for 1 minute.

2. Expel saline solution into a paper cup.

3. Swirl to mix cells in the cup and transfer 1 ml (1000 µl) of the liquid to 1.5 ml tube.

4. Place your sample tube, in a microcentrifuge, and spin for 1.5 minutes.

5. 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.

6. Resuspend cells in remaining saline by pipetting in and out.

7. Withdraw 30 µl of cell suspension, and add to tube containing 100 µl of Chelex. Vortex to mix.

8. Place tube in a thermal cycler for 10 minutes at 99°C.

9. After boiling, vortex tube. Place in a microcentrifuge and spin for 30 sec.

10. Transfer 30 µl of supernatant (containing the DNA) to clean 1.5 ml tube. Avoid cell debris and Chelex beads.

11. Store your sample on ice or in the freezer until ready to begin Step 12.

12.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.

13.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.

14.Label the cap of your tube with a number, as assigned by your teacher.

15.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°CExtending time and temperature 30 sec - 72°C Hold at -4°C (optional)

15.Use a micropipet with a fresh tip to add 15µl PCR sample/loading dye mixture into your assigned well of a 1% agarose gel.

16.Load 5 µl of the pBR322-BstNI size markers into one lane of gel.

17.Electrophorese at 130 volts for 20-30 minutes. Adequate separation will have occurred when the cresol red dye front has moved at least 50 mm from the wells.

18.Gel may be stained with 1 µg/ml ethidium bromide for 10 minutes followed by 20-30 minutes destaining with water.