analysis of gene expression using rt pcr
TRANSCRIPT
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Ammar ElakhdarDecember, 2015
Analysis of Gene Expression Using RT-PCR
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what is PCR?
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http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html
Polymerase Chain Reaction
DNA Denatures at 94°C
Primers anneal to single stranded DNA ~55°C
Thermostable TAQ polymerase extends primers at ~72°C
5’3’
5’ 3’
Target DNA is doubled.Cycle is then repeated.
Target DNA
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How was PCR discovered?
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PCR originates from DNAsequencing. So, lets first review DNA sequencing.
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Primer
3’5’
5’3’
Primer Anneals & DNA Polymerase Adds Deoxynucleoside triphosphates
37°CExtension
New DNA strandis created
Sequencing is performed by DNA replication
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ATGCATGCATGC????????????????????????????????????3’ 5’
TACGTACGTACGATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
3’DNAPol.
TACGTACGTACG????????????????????????????????ATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
3’
DNAPol.
dNTPs (or bases) are being added, butwe do not know the sequence.
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What if DNA extension could be terminatedat a known nucleotide using a mixture of
normal bases and termination bases
TACGTACGTACGTGTATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer DNA
Pol.
A
TACGTACGTACGTGT CGATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
ADNAPol.
A
Normal base getsincorporated
By probability termination will occurat every “A”
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dATPdGTPdCTPdTTP+
dATPdGTPdCTP+dTTP
dATPdGTP+dCTPdTTP
dATP+dGTPdCTPdTTP
A
T
GC
DNA
What if four reactions were set up tostop at each nucleotide?
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TACGTACGTACG ATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
DNAPol.
T
TACGTACGTACG GATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
T
DNAPol.
T
Normal base getsincorporated
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TACGTACGTACGT ATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
DNAPol.
G
TACGTACGTACGT TACATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
G
DNAPol.
G
Normal base getsincorporated
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TACGTACGTACGTGTAATGCATGCATGC????????????????????????????????????
3’ 5’
5’Primer
DNAPol.
C
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ATACGTACGTACG???5’
TACGTACGTACG??????5’ ATACGTACGTACG???????5’
TACGTACGTACG?????5’ G
GTACGTACGTACG?5’
TACGTACGTACG???????5’
CTACGTACGTACG????5’TACGTACGTACG???????5’
TACGTACGTACG??5’ T
TTACGTACGTACG5’TACGTACGTACG???????5’
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ATACGTACGTACG???5’
TACGTACGTACG??????5’ A
TACGTACGTACG?????5’ G
GTACGTACGTACG?5’
TACGTACGTACG??5’ T
TTACGTACGTACG5’
CTACGTACGTACG????5’
TACGTACGTACG???????5’
5’
3’
Chain-termination providessequence!
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dATPdGTPdCTPdTTP+ ddTTP
dATPdGTPdCTP+ ddCTPdTTP
dATPdGTP + ddGTPdCTPdTTP
dATP + ddATPdGTPdCTPdTTP
What causes chain termination?
Dideoxynucleoside Triphosphates
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Deoxynucleoside triphosphatesDeoxy adenosine triphosphate (dATP)Deoxy guanosine triphosphate (dGTP)Deoxy thymidine triphosphate (dTTP)Deoxy cytidine triphosphate (dCTP)
ChainTermination
3’
5’
DNAPolymerase
Lacks a 3’ hydroxyl group.Acts as a terminator because, once incorporated, no other nucelotide can be added.
X3’
5’
DNAPolymerase
Dideoxynucleoside triphosphatesDideoxy adenosine triphosphate (ddATP)Dideoxy guanosine triphosphate (ddGTP)Dideoxy thymidine triphosphate (ddTTP)Dideoxy cytidine triphosphate (ddCTP)
ChainExtension
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PhD 1943 Cambridge University
Nobel Prize In Chemistry 1958Amino acid sequence of insulin
Nobel Prize In Chemistry 1980Sequenced the first genome, phage Φ-X174, by hand using a method that he developed.Frederick Sanger
The Sanger Dideoxy sequencing methodwas the foundation for the discovery ofPCR.
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http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf
Dideoxy sequencing, one more time.
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Ok, but what is the connectionbetween DNA sequencing and
PCR?
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1983Emeryville, CaliforniaCetus Corporation
Henry Erlich was working on methods for detecting point mutations.
5’-TACGTACGTACGA*GGAGTCCGGAATG-3’
A? T? G? C?
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Why not do Sanger sequencing at a single base pair?
Kary B. Mullis
5’-TACGTACGTACGA*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’+
ddTTP ddCTPddGTPddATP
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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!
CC
TCA
GG
CC
TTAC
-5’
CCTCAGGCCTTAC-5’
CCTCAGGCCTTAC-5’
CCTCAGGCCTTAC-5’
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Kary and girlfriend chemist Jennifer BarnettMendocino County
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What if I use twoprimers for confirmation?
+
ddTTP ddCTPddGTPddATP
3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
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+
ddTTP ddCTPddGTPddATP
3’-ATGCATGCATGCTACCTCAGGCCTTAC-5’5’-GAATTCTACGTACGTACGATF-long
5’-TACGTACGTACGATGGAGTCCGGAATG-3’ ACCTCAGGCCTTAC-5’ R-short
F-long
R-short
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What about straynucleotide triphosphates?
+
ddTTP ddCTPddGTPddATP
3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
dNTP
dNTP
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I can destroy straydNTPs with alkalinephosphatase!
But, bacterial alkaline phosphatase will remain because it cannotbe heat killed. It will destroy theddNTP’s (not true).
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Second step to a Nobel Prize:
Make up problems thatdo not exist and try
to solve them.
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I can deplete nucleotidesby adding polymerasefirst without ddNTP’s
3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’
5’-GAATTCTACGTACGTACGAF-long
5’-TACGTACGTACGA*GGAGTCCGGAATG-3’
CCTCAGGCCTTAC-5’
R-short
dNTP
dNTP
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Denature and anneal primers
Polymerase extension
DNA replicated!
Anderson Valley
Third step to a Nobel Prize. Recognize PCR when
you find it.
1 Copy
2 Copies!
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NOBEL PRIZE!
…..not so fast
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Final step to a Nobel Prize:
Try to get someoneto listen to you.
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“…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.”
At first, people did not get it.
Then Joshua Lederberg (also a NobelLaureate) said:
“Why didn’t 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 SNP’s are associated withdisease. Do you have a risk allele?
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Let’s set up a PCR reactionand find out!
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Loci for Type 2 Diabetes and Triglyceride Levels
GCAGCTCACCTCCAGCTTTAGTTTTC[C/T]CATGACAGTAAGTCTATTACCCTCC
Risk allele
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First, you need to select primers for PCR
• No, you do not need a computer program to selectprimers.
• 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% G’s +C’s to ensurereasonable annealing temperature
That’s about it. Do not waste too much time selectingprimers.
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Forward Primer Selection
TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC
Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’
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TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC
Reverse Primer Selection
Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’
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AGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGC
Amplified Fragment Will Be 230bp
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Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’
Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’
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TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC
5’-AGAACACCATAACCTTTCCGGCCC-3’
Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’
Before ordering: Imagine the primers annealing to the DNA
Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’
AGCCACTGCACCCGACTGG-3’ Reverse Primer: 5’-GCGTG
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Order From IDTForward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’
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Start
Finish
H20 28.25μl10XPCR Buffer 5.0μl25mM MgCl2 3.0μl4mM dNTP’s 2.5μl10pmol Forward Primer 5.0μl (10pmol)10pmol Reverse Primer 5.0μl (10pmol)Template DNA >104 copies of target sequence (1.0μl)TAQ Polymerase 0.25μl (5u/μl)
PCR Components
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Use thin-wallPCR tubes
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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Mineral Oil
Sample
If your thermocycler does not have a hot bonnetor if you will need to open the hot bonnet to
remove or modify a reaction, use mineraloil.
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Runs on antifreeze and refrigerant
Paper towels to adsorb leakingorange-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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55°C
62°C61°C60°C59°C58°C57°C56°C
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 exceptit 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?
mRNA AAAAAAAAAAAA5’- -3’TTTTTTTTTTTT-5’
mRNA AAAAAAAAAAAA5’- -3’TTTTTTTTTTTT-5’cDNA
Reverse transcriptase Oligo (dT) Primer
Template For PCR
3’-
3’-
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RT-PCR measures the presenceof 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