PCR Lab Hershey High School Biotechnology Mr' Barber

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PCR LabHershey High School BiotechnologyMr.
Barber
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What is PCR?

• DNA replication gone crazy in a tube!
• Makes many copies of target sequence from
  template DNA
• Uses heat-resistant Taq polymerase from Thermus
  aquaticus

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Protocol Highlights

• Genomic DNA extraction
• InstaGene Chelex cation exchange resin
• binds cellular MgCl2
• 56oC loosens connective tissue and inactivates
  DNases
• 100oC ruptures cell membranes and denatures
  proteins

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InstaGene Extraction
Cell membrane
Nuclear membrane
Mg
Genomic DNA
Mg
Mg
Mg
Heat disrupts membranes
Mg
InstaGene matrix binds released cellular Mg
Mg
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Micropipet Use

• Twist dial to desired volume
• Pick up pipet tip
• Press plunger to first, soft stop
• Insert pipet tip into solution to be
  
• transferred
• Slowly release plunger to retrieve liquid
• Move pipet tip into desired tube
• Press plunger past first stop to second,
• hard stop to transfer liquid

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What is needed for PCR?

• Template (the DNA you want to amplify for the
  study)
• Sequence-specific primers flanking the target
  sequence
• Forward
• Reverse
• Nucleotides (As, Ts, Gs, Cs)
• Magnesium chloride (enzyme cofactor)
• Buffer, containing salt
• Taq polymerase

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How does PCR work?

• Heat (94oC) to denature DNA strands
• Cool (60oC) to anneal primers to template
• Warm (72oC) to activate Taq polymerase, which
  extends primers and replicates DNA
• Repeat multiple cycles

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Denaturing Template DNA
Heat causes DNA strands to separate
Denaturation of DNA at 94oC
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Annealing Primers

• Primers bind to the template sequence
• Taq polymerase recognizes double-stranded
  substrate

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5
Primers anneal at 60oC
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3
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Taq polymerase extends..

• Taq polymerase extends primer
• DNA is replicated

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Extend at 72oC
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Repeat denaturing, annealing, and extending 40
cycles
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The exact-length target product is made in the
third cycle
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Cycle 1
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Cycle 2
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Cycle 3
PCR Animation-1
PCR Animation-2
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The target sequence

• PV92 Alu insertion
• Found on chromosome 16

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PV92 Alu insertion

• A member of Alu repeat family
• Human-specific Alu insertion
• Found in a non-coding region of your DNA
• NOT diagnostic for any disease or disorder

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PCR Results

• The PV92 Alu is dimorphic so there are two
  possible PCR products
• 641 bp
• 941 bp

No insertion 641 bp
Alu insertion 941 bp
641 bp
300 bp Alu insert
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Actual Alu PCR Results
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Alu repeats

• Classified as SINEs (Short Interspersed
  Repetitive Element)
• Mobilized by an RNA polymerase-derived
  intermediate (retroposition)
• Approx. 500,000 Alu copies per haploid genome,
  representing about 5 of the genome
• Named for the Alu I restriction site within the
  element

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Evolutionary Significance of PV92 Alu Inserts

• Highly conserved
• Inserted in the last 1,000,000 years
• Genotypes (/, /-, -/-)
• Used in population genetics, paternity analysis,
  and forensics