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PCR

PCR. A technique to make a lot of DNA from only a little!. PCR. Full name of the process is Polymerase Chain Reaction Invented in 1983 by Dr. Kary Mullis Allows scientists to make unlimited copies of specific DNA fragments Can amplify DNA fragments of up to approximately 10,000 bp. PCR.

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PCR

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  1. PCR A technique to make a lot of DNA from only a little!

  2. PCR • Full name of the process is Polymerase Chain Reaction • Invented in 1983 by Dr. Kary Mullis • Allows scientists to make unlimited copies of specific DNA fragments • Can amplify DNA fragments of up to approximately 10,000 bp

  3. PCR Two problems exist for scientists to make a visual analysis of a specific DNA locus (site or location): • Must first isolate the specific locus from all the other millions of sites in chromosomes • Must amplify (duplicate) the locus so that you can see it on the gel

  4. PCR Requirements • Template DNA (what you extract) • Primers • DNA polymerase • DNTP (deoxyribonucleoside triphosphates) or the A’s, T’s, C’s, and G’s • Thermal Cycler

  5. PCR Requirements Primers • Short segments of DNA 20-30 bp long which “bracket” the desired DNA segment • One primer is a complement “forward” primer to produce DNA strand from left to right while one is a “reverse” primer that is for right to left strand A T T C G C G A A A T G T T G G G C A A C A G G T A C T C T A G C G C T T T AC C A G G T A C T C T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

  6. PCR Requirements • If primers are carefully chosen they can select a unique sequence in the genome

  7. PCR Requirements Heat stable DNA polymerase • Most commonly use Taq polymerase – • Thermus aquaticus (a bacteria found around hot springs) • Is an enzyme that helps form new bonds between the nucleotides in new strands of bacteria A T T C G C G A A A T G T T G G G C A A C A G G T A C T C T A G C G C T T T AC C A G G T A C T C T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

  8. PCR Requirements DNTP’s • Deoxyribonucleoside triphosphates – • Nitrogen bases: adenine, thymine, cytosine, and guanine • These DNTP’s attach to the exposed complementary bases of the original DNA A T T C G C G A A A T G T T G G G C A A C A G G T A C T C T A G C G C T T T AC A A C C C G T T G T C C A T G A G A T A T T C G C G A A A T G T T G G G C A A C A G G T A C T C T A A G C G C T T T A C A A C C C G T T G T C C A T G A G A T

  9. PCR Requirements Ready to go PCR Beads • For convenience, we use PCR beads which contain freeze-dried DNTP’s and Taq polymerase enzyme • All we add to them are the primer mix and template DNA **Note** These beads are veryexpensive – Almost $2.00 per bead!!! So don’t mess it up!!

  10. PCR: The Steps of the Process • 3 phases for each cycle (these vary slightly from one protocol to another) • Denaturing: (94-95°C) DNA strands separate into single strands • Annealing: (58°C) Primers anneal (attach) to the separated DNA strands • Extending: (72°C) New complementary strands are made as the Taq enzyme helps to form bonds with the DNTP’s

  11. PCR: The Steps of the Process • Approximately 30 cycles of these 3 phases are used • Each cycle produces twice as many targeted DNA segments as existed before • After 30 cycles approximately 1 billion copies are produced • Takes approximately 2 hours

  12. PCR Equipment: Thermal Cycler $2,600

  13. PCR Equipment: Thermal Cycler • It is possible to substitute 3 water baths at the 3 different temperatures for a thermal cycler, but… • DNA samples would have to be moved from water bath to water bath to water bath for 30 cycles!

  14. PCR Activity: Paper PCR • Use strips of paper to create 4 cycles of a PCR reaction. • Color code the reactions as follows: • Red: Template DNA strand (40 bp) • Write the following sequence on one red strip: A C G A T T G G G C C A A T A T A C G C G T G A T G T T C G A A G A G A C T

  15. PCR Activity: Paper PCR • Yellow: 1st copied strand • Blue: 2nd copied strands • Green: 3rd copied strands • Forward primers are white • Reverse primers are gray G A T T C C A T A

  16. Steps of DNA Isolation: • 10 mL of saline, swish, spit, use 1mL of this. • Centrifuged • Poured off saline & then resuspended • Removed 30 uL and added to 100 uL of Chelex to absorb cell debris and ions. • Boiled for 10 minutes. • Withdrew 30 uL & stored in new tube.

  17. Day 2: Amplification • Added 22.5 uL of Primer Mix into PCR bead. • Added 2.5 uL of DNA. • Quick spin to put everything together. • Thermal cycler • DAY 3: Ran gels

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