Transcript What does PCR stand for?

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?
To make many copies of a small section of DNA.
DNA Synthesis in vitro (in a test tube).
How does PCR work?
Very Similar to DNA Synthesis
How does PCR work?
http://www.lsic.ucla.edu/ls3/tutorials/gene_clonin
g.html
http://www.iupui.edu/~wellsctr/MMIA/htm/animati
ons.htm
DNALC
http://wps.prenhall.com/wps/media/objects/487/4
98929/CDA12_2/CDA12_2a/CDA12_2a.htm
What is needed for a PCR
reaction?
DNA template – DNA to be copied; “target
sequence”
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
reaction?
Taq Polymerase
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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
reaction?
Nucleotides – building
blocks of DNA
Thermalcycler –
computerized to
change temperatures
What are the 3 main steps of PCR?
Denaturing – DNA strands separate at hydrogen bonds
Temperature = 950C
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 PCR?
Extension – DNA is copied
Temperature = 720C
PCR Reaction Summary
1. Denaturing
2. Annealing
3. Extension
30 cycles
How many times can target DNA
be copied?
DNALC
What would someone do with
PCR?
Forensics
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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).
What would someone do with
PCR?
Medical (genotyping)
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Genetic diseases identified
pre- or post-natally.
Cancers identified and
classified.
Identifying bacterial or viral
strains for proper
treatment.
What would someone do with
PCR?
Food Science
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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.
What would someone do with
PCR?
Relationships
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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
BioRad “When You Need to Find
Out Who The Daddy Is”
BioRad “GTCA”
PCR Lab #1 - Alu
What DNA sequence are we going to
copy?
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Alu element
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Chromosome 16
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Does NOT code for a gene
PCR Lab #1 - Alu
95% of the human genome does NOT
code for proteins
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“Junk” DNA
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Nobody knows why we have it
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All of it is copied during DNA synthesis and
passed from one generation to the next
PCR Lab #1 - Alu
Alu is an example of a “jumping gene” (also
known as a transposon)
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Come from a virus infection
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All primates share the same initial Alu sequence
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Human chromosomes contain an estimated
1,000,000 Alu copies equaling 10% of the total
genome.
PCR Lab #1 - Alu
Alu elements are dimorphic (meaning two forms)
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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
What DNA sequence
are we going to copy?
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PTC taste receptor
gene (produces a
bitter taste)
PCR Lab #1 – PTC Taster
Chance discovery
PCR Lab #1 – PTC Taster
Being a “taster” is a dominant trait
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Homozygous dominant – TT
TASTERS
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Heterozygous – Tt
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Homozygous recessive - tt
NON-TASTERS
PCR Lab #1 – PTC Taster
About 70% of people are tasters
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58% for Aboriginal people of Australia
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98% of Native Americans
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.
PCR Lab #1 – PTC Taster
What will be our DNA source?
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Cheek cells
Rinse our mouths with a saline solution (0.8% NaCl)
PCR Lab #1 – PTC Taster
Why not just use pure water to rinse our
mouths?
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What would happen to our cells in 100% water?)
Due to osmosis, cells would burst before we could collect DNA
PCR Lab #1 – PTC Taster
Cells are collected, allowed to settle, and
then mixed with Chelex solution.
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Negatively charged beads – removes Mg2+
which inhibits Taq polymerase.
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.
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
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’
Taq Pol
Anneal;
Extend; ~60
7200CC
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’
PCR Lab #2 - mtDNA
What DNA
sequence are we
going to copy?
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mtDNA control
element
Does NOT code
for a gene
PCR Lab #2 - mtDNA
In which organelle is this DNA stored?
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mitochondria
All DNA is NOT stored
in nucleus!
PCR Lab #2 - mtDNA
How many genes
are on this DNA?
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37 genes in
16,569bp
PCR Lab #2 - mtDNA
What proteins do
these genes make?
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Proteins involved in
cellular respiration
PCR Lab #2 - mtDNA
Interesting facts
regarding mtDNA
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Always inherited from
mom
Mutates at a constant
rate
PCR Lab #2 - mtDNA
PCR Lab #2 - mtDNA
Compare
sequences to
determine
genetic
relationships
(phylogenetic
tree)
PCR Lab #2 - mtDNA
Interesting facts
regarding mtDNA
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Mitochondria may
have been a bacterial
cell at some point