Brad Porter

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Lecture Introduction to PCR Analysis of
Gene Expression Using RT-PCR
Fri, June 15, 2007 1100 1150 AM 
Brad Porter
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Briefly, what is PCR?
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Polymerase Chain Reaction
Target DNA
DNA Denatures at 94C
http//www.sumanasinc.com/webcontent/anisamples/mo
lecularbiology/pcr.html
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How was PCR discovered?
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PCR originates from DNA sequencing. So, lets
first review DNA sequencing.
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Sequencing is performed by DNA replication
37C
Extension
New DNA strand is created
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dNTPs (or bases) are being added, but we do not
know the sequence.
TACGTACGTACG????????????????????????????????
5
3
Primer
DNA Pol.
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What if DNA extension could be terminated at a
known nucleotide using a mixture of normal bases
and termination bases
TACGTACGTACGTGT
5
A
Primer
By probability termination will occur at every A
TACGTACGTACGTGT CG
A
5
Primer
Normal base gets incorporated
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What if four reactions were set up to stop at
each nucleotide?
DNA
dATP dGTP dCTP dTTP
dATP dGTP dCTP dTTP
dATP dGTP dCTP dTTP
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TACGTACGTACG
5
Primer
TACGTACGTACG G
T
5
Primer
Normal base gets incorporated
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TACGTACGTACGT
5
Primer
G
TACGTACGTACGT TAC
5
Primer
Normal base gets incorporated
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TACGTACGTACGTGTA
5
Primer
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TACGTACGTACG???
5
TACGTACGTACG?
5
TACGTACGTACG??
5
TACGTACGTACG????
5
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Chain-termination provides sequence!
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TACGTACGTACG????
5
5
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What causes chain termination?
Dideoxynucleoside Triphosphates
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Chain Extension
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DNA Polymerase
Deoxynucleoside triphosphates Deoxy adenosine
triphosphate (dATP) Deoxy guanosine triphosphate
(dGTP) Deoxy thymidine triphosphate (dTTP) Deoxy
cytidine triphosphate (dCTP)
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Chain Termination
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DNA Polymerase
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X
Lacks a 3 hydroxyl group. Acts as a terminator
because, once incorporated, no other nucelotide
can be added.
Dideoxynucleoside triphosphates Dideoxy adenosine
triphosphate (ddATP) Dideoxy guanosine
triphosphate (ddGTP) Dideoxy thymidine
triphosphate (ddTTP) Dideoxy cytidine
triphosphate (ddCTP)
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The Sanger Dideoxy sequencing method was the
foundation for the discovery of PCR.
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Dideoxy sequencing, one more time.
http//smcg.cifn.unam.mx/enp-unam/03-EstructuraDel
Genoma/animaciones/secuencia.swf
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Ok, but what is the connection between DNA
sequencing and PCR?
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1983 Emeryville, California Cetus Corporation
Henry Erlich was working on methods for
detecting point mutations.
5-TACGTACGTACGAGGAGTCCGGAATG-3
A?
T?
G? C?
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5-TACGTACGTACGAGGAGTCCGGAATG-3
CCTCAGGCCTTAC-5

Kary B. Mullis
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First step to a Nobel Prize As you think,
ignore obvious problems.
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Kary wanted to use total genomic DNA, but he
forgot the primer would likely mis-pair and ruin
his experiment. In misguided puttering, Kary
kept thinking!
CCTCAGGCCTTAC-5
CCTCAGGCCTTAC-5
CCTCAGGCCTTAC-5
CCTCAGGCCTTAC-5
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Kary and girlfriend chemist Jennifer Barnett
Mendocino County
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5-TACGTACGTACGATGGAGTCCGGAATG-3
ACCTCAGGCCTTAC-5
R-short
F-long
5-GAATTCTACGTACGTACGAT
3-ATGCATGCATGCTACCTCAGGCCTTAC-5

F-long
R-short
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dNTP
5-TACGTACGTACGAGGAGTCCGGAATG-3
CCTCAGGCCTTAC-5
R-short
dNTP

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I can destroy stray dNTPs with alkaline phosphatas
e!
But, bacterial alkaline phosphatase will remain
because it cannot be heat killed. It will destroy
the ddNTPs (not true).
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Second step to a Nobel Prize Make up problems
that do not exist and try to solve them.
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I can deplete nucleotides by adding
polymerase first without ddNTPs
dNTP
dNTP
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Third step to a Nobel Prize. Recognize PCR when
you find it.
Anderson Valley
1 Copy
Denature and anneal primers
Polymerase extension
2 Copies!
DNA replicated!
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NOBEL PRIZE! ..not so fast
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Final step to a Nobel Prize Try to get
someone to listen to you.
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At first, people did not get it.
no one was particularly enthusiastic about
it. 1984 annual Cetus Scientific
Meeting..nobody seemed to be interested in my
poster. People would glance at it and keep
walking.
Then Joshua Lederberg (also a Nobel Laureate)
said Why didnt I think of that?
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1993 Nobel Prize
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So, how is PCR important to your life, right
now? Many SNPs are associated with disease. Do
you have a risk allele?
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Lets set up a PCR reaction and find out!
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Loci for Type 2 Diabetes and Triglyceride Levels
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First, you need to select primers for PCR
No, you do not need a computer program to
select primers. I prefer 24 bp long and end on
G or C Others prefer 20 bp long and end on A
or T Try to have at least 50 Gs Cs to
ensure reasonable annealing temperature Thats
about it. Do not waste too much time
selecting primers.
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Forward Primer Selection
TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAA
AAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAG
GTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACA
GATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCT
GTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACT
TAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATG
AAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGAC
TTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAAC
CTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGC
TCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGA
TCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTAC
TGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCG
GGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGG
GTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGA
AACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAAT
CCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCG
GAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACA
AGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAA
GAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACA
CAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGC
CCCTTAAC
Forward Primer 5-AGAACACCATAACCTTTCCGGCCC-3
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Reverse Primer Selection
TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAA
AAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAG
GTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACA
GATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCT
GTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACT
TAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATG
AAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGAC
TTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAAC
CTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGC
TCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGA
TCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTAC
TGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCG
GGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGG
GTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGA
AACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAAT
CCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCG
GAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACA
AGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAA
GAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACA
CAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGC
CCCTTAAC
Reverse Primer 5-GCGTGAGCCACTGCACCCGACTGG-3
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Amplified Fragment Will Be 230bp
AGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTG
CCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCT
ATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGG
TTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAA
GGTTTTCCAGTCGGGTGCAGTGGCTCACGC
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Forward Primer 5-AGAACACCATAACCTTTCCGGCCC-3
Reverse Primer 5-GCGTGAGCCACTGCACCCGACTGG-3
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Before ordering Imagine the primers annealing to
the DNA
Forward Primer 5-AGAACACCATAACCTTTCCGGCCC-3
AGCCACTGCACCCGACTGG-3?
Reverse Primer 5-GCGTG
Reverse Primer 5-GCGTGAGCCACTGCACCCGACTGG-3
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Order From IDT
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PCR Components
H20 28.25µl 10XPCR Buffer 5.0µl 25mM MgCl2
3.0µl 4mM dNTPs 2.5µl 10pmol Forward Primer
5.0µl (10pmol) 10pmol Reverse Primer 5.0µl
(10pmol) Template DNA gt104 copies of target
sequence (1.0µl) TAQ Polymerase 0.25µl (5u/µl)
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Use thin-wall tubes designed for PCR
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Old School Perkin Elmer 2400 PCR Thermocycler
Hot bonnet prevents condensation.
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If your thermocycler does not have a hot
bonnet or if you will need to open the hot bonnet
to remove or modify a reaction, use mineral oil.
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Runs on antifreeze and refrigerant
Paper towels to adsorb leaking orange-colored
antifreeze.
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Antifreeze
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R134a frigerant
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Radiator fan
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New Thermocyclers Peltier Cooling Gradient
Blocks
Peltier effect It occurs when a current is
passed through two dissimilar metals or
semiconductors (n-type and p-type) that are
connected to each other at two junctions (Peltier
junctions). The current drives a transfer of heat
from one junction to the other one junction
cools off while the other heats up as a result,
the effect is often used for thermoelectric
cooling. This effect was observed in 1834 by Jean
Peltier.
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A typical PCR thermocycling program
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Gradient thermocyclers allow for optimization of
the annealing temperature
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What is RT-PCR? Reverse Transcription-
Polymerase Chain Reaction
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RT-PCR is like any other PCR except it uses cDNA
as a template.
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How do you make cDNA?
cDNA can be created from RNA using RNA-dependent
DNA polymerase (reverse transcriptase)
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How do you make cDNA?
Reverse transcriptase
Oligo (dT) Primer
3-
3-
TTTTTTTTTTTT
-5
cDNA
AAAAAAAAAAAA
5-
-3
mRNA
Template For PCR
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RT-PCR measures the presence of cDNA
corresponding to its respective RNA. RT-PCR
is, therefore, used to indirectly estimate RNA
abundance which MAY indicate the level of gene
expression.
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Major Points
Sequencing and PCR both use DNA polymerase and
replicate DNA (PCR uses TAQ DNA
polymerase). Kary Mullis discovered PCR while
thinking about a possible dideoxy sequencing
experiment. Half of a Nobel discovery is
finding it, the other half is realizing what
you have found. RT-PCR is used to estimate
gene expression