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Detection of the human Mitochondrial DNA

Detection of the human Mitochondrial DNA. A Polymerase Chain Reaction Experiment. Polymerase Chain Reaction: another method of DNA amplification. Basis of PCR. Create conditions “in vitro” for DNA replication. Requirements for replication. DNA template Primers Taq DNA Polymerase**

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Detection of the human Mitochondrial DNA

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  1. Detection of the human Mitochondrial DNA A Polymerase Chain Reaction Experiment

  2. Polymerase Chain Reaction: another method of DNA amplification

  3. Basis of PCR • Create conditions “in vitro” for DNA replication.

  4. Requirements for replication • DNA template • Primers • Taq DNA Polymerase** • Nucleotides • MgCl++

  5. Unwinding DNA Annealing Extension 98C 45-60C 72C Temperature cycles plays a key role: http://www.dnalc.org/Shockwave/pcranwhole.html

  6. Primers determine • The sequence of DNA that will be amplified.

  7. Basis for sequence specific amplification • Two primers are used to bracket the area you want to amplify. • Primers are single stranded synthetic sequences of DNA normally 20-30 bp. • One primer is complementary to the beginning of the target gene on one strand while the other primer is complementary to end of the target gene on the complementary strand.

  8. Unwinding DNA Annealing Extension 98C 45-60C 72C Summary: http://www.dnalc.org/Shockwave/pcranwhole.html

  9. The experiment is divided into 2 parts • Isolate human DNA from cheek cells • The DNA will be amplified using PCR • The DNA will be analyzed using gel electrophoresis

  10. Mitochondrial DNA • Circular • 16,569 bp • 37 genes • Encodes 13 polypeptides

  11. Mitochondria DNA Proteins of electron transport system

  12. ISOLATION OF DNA AND PCR PROTOCOL

  13. FLOW CHART • Isolation cheek cell DNA • Amplify using PCR • Identify PCR-DNA product by gel electrophoresis

  14. Each person should obtain • Cotton swabs • A test tube with 2 mls of buffer • A screw top microfuge tube • 2 transfer pipets

  15. Isolation of DNA • Cheek Cells • Obtain cells by swabbing the inside of the mouth with a cotton tipped applicator.

  16. Next… • Place cotton head in 2 ml of PBS (in conical tube) • Swirl vigorously for 1 min. to dislodge cells • Press the cotton head against the walls to squeeze out as much liquid as possible • Use new applicator and repeat the above steps.

  17. Next… • Transfer 2 ml to screw top tube. • Spin at 5000 rpm for 5 min. Be sure you have pellet. If not repeat swabbing. • Remove supernantant but SAVE PELLET (these are your cells!) • Add 100 ul of chelating agent to sample

  18. Next… • Re-suspend the pellet in 100 ul of chelator solution: MAKE SURE PELLET IS RESUSPENDED! • Place screw top microfuge tube in boiling waterbath for 10 minutes to break (lyse) cells.

  19. Next: • After 10 min boiling allow tube to cool (2 minutes and then spin microfuge tube for 2 minutes (5000 rpm)

  20. Next: • Obtain a PCR tube with “PCR bead” • Add 20 ul primers to bead and 5 ul of your supernatant and gently mix. • Label your PCR tube your assigned seat number • Place in PCR!

  21. Summary of PCR tube • To PCR tube containing “bead” add: • 20.0 ul of primer solution • 5.0 ul of cheek cell DNA

  22. 94C 55C 72C 60 seconds 60 seconds 60 seconds Mt DNA : PCR reaction 30 cycles at: Initial denaturation at 94C for 4 minutes Final Extension: 72C for 5 minutes EXPECTED FRAGMENT SIZES: 921 and 672

  23. The gel electrophoresis • Analysis of your results will be carried and a photo taken.

  24. After PCR • Gel electrophoresis • Warm samples (including ladder) 2 minutes at 50 C • Load 25 ul of the sample • Load 25 ul of ladder

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