Polymerase Chain Reaction

1
Polymerase Chain Reaction

• a.k.a. Howd they get all that DNA from just a
  little blood?

2
What does PCR stand for?

• Polymerase Chain Reaction
• Developed by Kary Mullis Nobel Prize
• Received a 20,000 bonus later sold it to
  Hoffman-LaRoche for 300,000,000.

3
What is the goal of PCR?

• To make many copies of a small section of DNA.
• DNA Synthesis in vitro (in a test tube).

4
How does PCR work?

• Very Similar to DNA Synthesis

5
How does PCR work?

• http//www.lsic.ucla.edu/ls3/tutorials/gene_clonin
  g.html
• http//www.iupui.edu/wellsctr/MMIA/htm/animations
  .htm
• DNALC
• http//wps.prenhall.com/wps/media/objects/487/4989
  29/CDA12_2/CDA12_2a/CDA12_2a.htm

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What is needed for a PCR reaction?

• DNA template DNA to be copied target sequence

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What is needed for a PCR reaction?

• PCR primers short DNA sequences that bind to
  DNA are complimentary to beginning and end of
  target sequence

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What is needed for a PCR reaction?

• Taq Polymerase
• Polymerase copies DNA
• From heat loving bacteria - Thermus aquaticus.
• Can survive hot temperatures needed in PCR
  reaction.
• Always copies in 5 ? 3 direction

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What is needed for a PCR reaction?

• Nucleotides building blocks of DNA
• Thermalcycler computerized to change
  temperatures

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What are the 3 main steps of PCR?

• Denaturing DNA strands separate at hydrogen
  bonds
• Temperature 950C

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What are the 3 main steps of PCR?

• Annealing Primers bind
• Temperature is dependent on primer sequence
• Annealing temperature 3(GC) 2(AT)
• of H-bonds between nucleotides

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What are the 3 main steps of PCR?

• Extension DNA is copied
• Temperature 720C

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PCR Reaction Summary
1. Denaturing 2. Annealing 3. Extension
30 cycles
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How many times can target DNA be copied?

• DNALC

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What would someone do with PCR?

• Forensics
• Identification of corpses or body parts.
• Analysis of pathogens.
• Eliminate or link suspects to scene of crime
  through a sample (blood, human hair, skin, semen)
  left at scene or circumstantial links (pet hair,
  plant parts).

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What would someone do with PCR?

• Medical (genotyping)
• Genetic diseases identified pre- or post-natally.
• Cancers identified and classified.
• Identifying bacterial or viral strains for proper
  treatment.

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What would someone do with PCR?

• Food Science
• Samples can be tested for genetic engineering
  traits.
• Bacterial contaminants and source of
  contamination can be quickly identified.
• Pedigrees and traits of valuable animal food
  stocks can be verified.

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What would someone do with PCR?

• Relationships
• Paternity.
• Evolution developing relationships between
  organisms or studying extinct species from museum
  specimens.
• Ecology tracking organisms or defining
  biodiversity by identifying unique species.

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PCR Review Music Video
BioRad When You Need to Find Out Who The Daddy
Is
BioRad GTCA
20
PCR Lab 1 - Alu

• What DNA sequence are we going to copy?
• Alu element
• Chromosome 16
• Does NOT code for a gene

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PCR Lab 1 - Alu

• 95 of the human genome does NOT code for
  proteins
• Junk DNA
• Nobody knows why we have it
• All of it is copied during DNA synthesis and
  passed from one generation to the next

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PCR Lab 1 - Alu

• Alu is an example of a jumping gene (also known
  as a transposon)
• Come from a virus infection
• All primates share the same initial Alu sequence
• Human chromosomes contain an estimated 1,000,000
  Alu copies equaling 10 of the total genome.

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PCR Lab 1 - Alu

• Alu elements are dimorphic (meaning two forms)
• Chromosome 16 either has this Alu element or it
  doesnt
• Since chromosomes come in pairs, a person is
  either , -, or -- .
• DNA sequences such as Alu are used in diagnosis
  of genetic disease, forensic identification, and
  paternity testing.

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PCR Lab 1 PTC Taster

• What DNA sequence are we going to copy?
• PTC taste receptor gene (produces a bitter taste)

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PCR Lab 1 PTC Taster

• Chance discovery

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PCR Lab 1 PTC Taster

• Being a taster is a dominant trait
• Homozygous dominant TT
• Heterozygous Tt
• Homozygous recessive - tt

TASTERS
NON-TASTERS
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PCR Lab 1 PTC Taster

• About 70 of people are tasters
• 58 for Aboriginal people of Australia
• 98 of Native Americans

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PCR Lab 1 PTC Taster

• Non-taster PCR product -

221 bp

• Taster PCR product -

221 bp

• SNP Single Nucleotide Polymorphism. One
  letter makes a big difference.

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PCR Lab 1 PTC Taster

• What will be our DNA source?
• Cheek cells
• Rinse our mouths with a saline solution - (0.8
  NaCl)

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PCR Lab 1 PTC Taster

• Why not just use pure water to rinse our mouths?
• What would happen to our cells in 100 water?)

Due to osmosis, cells would burst before we could
collect DNA
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PCR Lab 1 PTC Taster

• Cells are collected, allowed to settle, and then
  mixed with Chelex solution.
• Negatively charged beads removes Mg2 which
  inhibits Taq polymerase.

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PCR Lab 1 PTC Taster

• Chelex, cheek cell mixture is heated to 980C.
  Heat bursts cells open and cell debris is bound
  to Chelex beads.
• Chelex beads and cell debris is heavy so it will
  settle to bottom of tube.
• We will collect the supernatant (contains DNA)
  and will freeze it.
• Set up PCR reaction on Thursday.

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3 A T C G G A C C G A C T G C A T G C C A G T T
A A T A G C C A A T 5
5 T A G C C T G G C T G A C G T A C G G T C A A
T T A T C G G T T A 3
Denature 950C
Taq Pol
3 A T C G G A C C G A C T G C A T G C C A G T T
A A T A G C C A A T 5
Anneal 600C
Extend 720C
5 C T G G 3
3 T A G C 5
Taq Pol
5 T A G C C T G G C T G A C G T A C G G T C A A
T T A T C G G T T A 3
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PCR Lab 2 - mtDNA

• What DNA sequence are we going to copy?
• mtDNA control element
• Does NOT code for a gene

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PCR Lab 2 - mtDNA

• In which organelle is this DNA stored?
• mitochondria
• All DNA is NOT stored
• in nucleus!

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PCR Lab 2 - mtDNA

• How many genes are on this DNA?
• 37 genes in 16,569bp

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PCR Lab 2 - mtDNA

• What proteins do these genes make?
• Proteins involved in cellular respiration

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PCR Lab 2 - mtDNA

• Interesting facts regarding mtDNA
• Always inherited from mom
• Mutates at a constant rate

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PCR Lab 2 - mtDNA
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PCR Lab 2 - mtDNA

• Compare sequences to determine genetic
  relationships (phylogenetic tree)

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PCR Lab 2 - mtDNA

• Interesting facts regarding mtDNA
• Mitochondria may have been a bacterial cell at
  some point