Detection of the human VNTR using PCR*

1. Detection of the human VNTR using PCR* *A Polymerase Chain Reaction Experiment

2. 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

3. Polymerase Chain Reaction: another method of DNA amplification

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

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

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

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

9. 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.

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

12. 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

13. Highlights of Key Steps of your DNA Isolation

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 1 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 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 65C 72C 30 seconds 30 seconds 30 seconds VNTR : PCR reaction 32 cycles at:

23. The gel electrophoresis • Analysis of your results will be carried and provided to you (time may not be available at the next lab for gel electrophoresis).

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