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Polymerase Chain Reaction. a.k.a. “How’d they get all that DNA from just a little blood?”. 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. What is the goal of PCR?.

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polymerase chain reaction

Polymerase Chain Reaction

a.k.a. “How’d they get all that DNA from just a little blood?”

what does pcr stand for
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.
what is the goal of pcr
What is the goal of PCR?
  • To make many copies of a small section of DNA.
  • DNA Synthesis in vitro (in a test tube).
how does pcr work
How does PCR work?
  • Very Similar to DNA Synthesis
how does pcr work1
How does PCR work?
  • http://www.lsic.ucla.edu/ls3/tutorials/gene_cloning.html
  • http://www.iupui.edu/~wellsctr/MMIA/htm/animations.htm
  • DNALC
  • http://wps.prenhall.com/wps/media/objects/487/498929/CDA12_2/CDA12_2a/CDA12_2a.htm
what is needed for a pcr reaction
What is needed for a PCR reaction?
  • DNA template – DNA to be copied; “target sequence”
what is needed for a pcr reaction1
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
what is needed for a pcr reaction2
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
what is needed for a pcr reaction3
What is needed for a PCR reaction?
  • Nucleotides – building blocks of DNA
  • Thermalcycler – computerized to change temperatures
what are the 3 main steps of pcr
What are the 3 main steps of PCR?
  • Denaturing – DNA strands separate at hydrogen bonds
  • Temperature = 950C
what are the 3 main steps of pcr1
What are the 3 main steps of PCR?
  • Annealing – Primers bind
  • Temperature is dependent on primer sequence
  • Annealing temperature = 3(G+C) + 2(A+T)
  • # of H-bonds between nucleotides
what are the 3 main steps of pcr2
What are the 3 main steps of PCR?
  • Extension – DNA is copied
  • Temperature = 720C
pcr reaction summary
PCR Reaction Summary

1. Denaturing

2. Annealing

3. Extension

30 cycles

what would someone do with pcr
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).
slide16
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.
slide17
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.
slide18
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.
pcr review music video
PCR Review Music Video

BioRad “When You Need to Find Out Who The Daddy Is”

BioRad “GTCA”

pcr lab 1 alu
PCR Lab #1 - Alu
  • What DNA sequence are we going to copy?
    • Alu element
    • Chromosome 16
    • Does NOT code for a gene
pcr lab 1 alu1
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
pcr lab 1 alu2
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.
pcr lab 1 alu3
PCR Lab #1 - Alu
  • Alu elements are dimorphic (meaning two forms)
    • Chromosome 16 either has this Alu element or it doesn’t
    • 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.
pcr lab 1 ptc taster
PCR Lab #1 – PTC Taster
  • What DNA sequence are we going to copy?
    • PTC taste receptor gene (produces a bitter taste)
pcr lab 1 ptc taster1
PCR Lab #1 – PTC Taster
  • Being a “taster” is a dominant trait
    • Homozygous dominant – TT
    • Heterozygous – Tt
    • Homozygous recessive - tt

TASTERS

NON-TASTERS

slide27
PCR Lab #1 – PTC Taster
  • About 70% of people are tasters
    • 58% for Aboriginal people of Australia
    • 98% of Native Americans
pcr lab 1 ptc taster2
PCR Lab #1 – PTC Taster

221 bp

  • Non-taster PCR product -

Taster PCR product -

221 bp

SNP – Single Nucleotide Polymorphism. One “letter” makes a big difference.

pcr lab 1 ptc taster3
PCR Lab #1 – PTC Taster
  • What will be our DNA source?
    • Cheek cells
    • Rinse our mouths with a saline solution - (0.8% NaCl)
pcr lab 1 ptc taster4
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

pcr lab 1 ptc taster5
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.
pcr lab 1 ptc taster6
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.
slide33
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’

slide34
PCR Lab #2 - mtDNA
  • What DNA sequence are we going to copy?
    • mtDNA control element
    • Does NOT code for a gene
slide35
PCR Lab #2 - mtDNA
  • In which organelle is this DNA stored?
    • mitochondria
    • All DNA is NOT stored

in nucleus!

slide36
PCR Lab #2 - mtDNA
  • How many genes are on this DNA?
    • 37 genes in 16,569bp
slide37
PCR Lab #2 - mtDNA
  • What proteins do these genes make?
    • Proteins involved in cellular respiration
slide38
PCR Lab #2 - mtDNA
  • Interesting facts regarding mtDNA
    • Always inherited from mom
    • Mutates at a constant rate
slide40
PCR Lab #2 - mtDNA
  • Compare sequences to determine genetic relationships (phylogenetic tree)
slide41
PCR Lab #2 - mtDNA
  • Interesting facts regarding mtDNA
    • Mitochondria may have been a bacterial cell at some point
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