PCR

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PCR

• A technique to make a lot of DNA from only a
  little!

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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

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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

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PCR Requirements

1. Template DNA (what you extract)
2. Primers
3. DNA polymerase
4. DNTP (deoxyribonucleoside triphosphates) or the
   As, Ts, Cs, and Gs
5. Thermal Cycler

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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 A C
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
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PCR Requirements

• If primers are carefully chosen they can select a
  unique sequence in the genome

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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 A C
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
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PCR Requirements

• DNTPs
• Deoxyribonucleoside triphosphates
• Nitrogen bases adenine, thymine, cytosine, and
  guanine
• These DNTPs 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 A C
A A C C C G T T G T C C A T G A G A T
C A G G T A C T C
A T T C G C G A A A T G T T G G G C A A
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
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PCR Requirements

• Ready to go PCR Beads
• For convenience, we use PCR beads which contain
  freeze-dried DNTPs and Taq polymerase enzyme
• All we add to them are the primer mix and
  template DNA
• Note
• These beads are very expensive Almost 2.00 per
  bead!!! So dont mess it up!!

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PCR The Steps of the Process

• 3 phases for each cycle (these vary slightly from
  one protocol to another)
• Denaturing (94-95C) DNA strands separate into
  single strands
• Annealing (58C) Primers anneal (attach) to the
  separated DNA strands
• Extending (72C) New complementary strands are
  made as the Taq enzyme helps to form bonds with
  the DNTPs

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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

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PCR Equipment Thermal Cycler
2,600
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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!

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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

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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
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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.

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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