Transcript Mitochondrial DNA - Winona Senior High School

Mitochondrial DNA
Sequencing DNA from Human Cheek
Cells Using PCR
General Overview
• Day 1: Collect and wash cheek cells
• Day 2: PCR(Polymerase Chain Reaction)
• Day 3: Gel Electrophoresis and Prepare
for sequencing
PCR
• 1. Denature(95°C): breaks DNA strands
apart by separating
hydrogen bonds
• 2. Anneal(50-65°C): Primer begins adding
nucleotides
• 3. Extension(72°C): Replicates DNA
• 25-50 cycles run to amplify DNA(each
doubles the DNA)
cycle
Primers and Enzymes
• Primer is the starting point to start
polymerase amplifying the DNA
• Taq (Thermus aquaticus): bacteria from hot
springs contains enzymes that can withstand
the high temperatures of PCR
Gel Electrophoresis
• Verify if product is at
440 base pairs(bp)
Sequencing DNA
Final Sequence Printout
Interesting Facts
• Mitochondrial DNA is from the mother
• Father DNA is destroyed during
development
• Inbreeding causes defects to occur over
multiple generations
• 5’ reads left to right(normal sequence
reading)
• 3’ reads right to left
• Primers are single stranded DNA
• EXOSAP will chew up excess primers and
nucleotides. This makes for a cleaner
sequencing of DNA