Introduction to PCR and analysis of gene expression using RT-PCR

Brad Porter

Lecture:

Introduction to PCR & Analysis of Gene Expression Using RT-PCR

Fri, June 15, 200711:00 – 11:50 AM

Briefly, what is PCR?

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

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

How was PCR discovered?

PCR originates from DNAsequencing. So, lets first review DNA sequencing.

Primer

3’5’

5’3’

Primer Anneals & DNA Polymerase Adds Deoxynucleoside triphosphates

37°C

Extension

New DNA strandis created

Sequencing is performed by DNA replication

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.

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

A

DNAPol.

A

Normal base getsincorporated

By probability termination will occurat every “A”

dATPdGTPdCTPdTTP+

dATPdGTPdCTP+dTTP

dATPdGTP+dCTPdTTP

dATP+dGTPdCTPdTTP

A

T

GC

DNA

What if four reactions were set up tostop at each nucleotide?

TACGTACGTACG ATGCATGCATGC????????????????????????????????????

3’ 5’

5’Primer

DNAPol.

T

TACGTACGTACG GATGCATGCATGC????????????????????????????????????

3’ 5’

5’Primer

T

DNAPol.

T


TACGTACGTACGT ATGCATGCATGC????????????????????????????????????

3’ 5’

5’Primer

DNAPol.

G

TACGTACGTACGT TACATGCATGCATGC????????????????????????????????????

3’ 5’

5’Primer

G

DNAPol.

G


TACGTACGTACGTGTAATGCATGCATGC????????????????????????????????????

3’ 5’

5’Primer

DNAPol.

C

ATACGTACGTACG???5’

TACGTACGTACG??????5’ A

TACGTACGTACG???????5’

TACGTACGTACG?????5’ G

GTACGTACGTACG?5’


CTACGTACGTACG????5’


TACGTACGTACG??5’ T

TTACGTACGTACG5’


ATACGTACGTACG???5’

TACGTACGTACG??????5’ A

TACGTACGTACG?????5’ G

GTACGTACGTACG?5’

TACGTACGTACG??5’ T

TTACGTACGTACG5’

CTACGTACGTACG????5’


5’

3’

Chain-termination providessequence!

dATPdGTPdCTPdTTP+ ddTTP

dATPdGTPdCTP+ ddCTP

dTTP

dATPdGTP + ddGTP

dCTPdTTP

dATP + ddATP

dGTPdCTPdTTP

What causes chain termination?

Dideoxynucleoside Triphosphates

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

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.

http://en.wikipedia.org/wiki/Image:Frederick_Sanger2.jpg

http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/animaciones/secuencia.swf

Dideoxy sequencing, one more time.

http://smcg.cifn.unam.mx/enp-unam/03-EstructuraDelGenoma/

Ok, but what is the connectionbetween DNA sequencing and

PCR?

1983Emeryville, CaliforniaCetus Corporation

Henry Erlich was working on methods for detecting point mutations.

5’-TACGTACGTACGA*GGAGTCCGGAATG-3’

A? T? G? C?

Why not do Sanger sequencing at a single base pair?

Kary B. Mullis


CCTCAGGCCTTAC-5’+

ddTTP ddCTPddGTPddATP

First step to a Nobel Prize:

As you think, ignore obvious problems.

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

TC

AG

GC

CT

TA

C-5’

CCTCAGGCCTTAC-5’

CCTCAGGCCTTAC-5’

CCTCAGGCCTTAC-5’

Kary and girlfriend chemist Jennifer BarnettMendocino County

What if I use twoprimers for confirmation?

+


3’-ATGCATGCATGCT*CCTCAGGCCTTAC-5’

5’-GAATTCTACGTACGTACGAF-long


CCTCAGGCCTTAC-5’

R-short

+


3’-ATGCATGCATGCTACCTCAGGCCTTAC-5’5’-GAATTCTACGTACGTACGATF-long

5’-TACGTACGTACGATGGAGTCCGGAATG-3’ ACCTCAGGCCTTAC-5’ R-short

F-long

R-short

What about straynucleotide triphosphates?

+





CCTCAGGCCTTAC-5’

R-short

dNTP

dNTP

I can destroy straydNTPs with alkalinephosphatase!

But, bacterial alkaline phosphatase will remain because it cannotbe heat killed. It will destroy theddNTP’s (not true).

Second step to a Nobel Prize:

Make up problems thatdo not exist and try

to solve them.

I can deplete nucleotidesby adding polymerasefirst without ddNTP’s




CCTCAGGCCTTAC-5’

R-short

dNTP

dNTP

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!

NOBEL PRIZE!

…..not so fast

Final step to a Nobel Prize:

Try to get someoneto listen to you.

“…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?”

1993 Nobel Prize

So, how is PCR important to your life,right now?

Many SNP’s are associated withdisease. Do you have a risk allele?

Let’s set up a PCR reactionand find out!

Loci for Type 2 Diabetes and Triglyceride Levels

GCAGCTCACCTCCAGCTTTAGTTTTC[C/T]CATGACAGTAAGTCTATTACCCTCC

Risk allele

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.

Forward Primer Selection

TAAATTCTTTGGAACAGGGGCATGGATTATAAAAGATGTAAGATAATAAAAAGCATTTGTATTTGACTTTGGAATGTATTGTACTTACATTTGTCTAGAGGTGTGTCTATTCTGGCTATTCTCTTTAAAGGAGCCATTCTATCGTGAACAGATCCTGTTGGAGCTGTTTTCTTGTTCTACCAACCTTCAGCCACCTCTCTGTCTTTCATATTACTTATTGGCAGGGTTTCAAAAGGTTTTAGTCCTTACTTAATATAAACAAAAATGTACAATATTGACAAAGTTTCAGTTAAGCAGATGAAATTCTAAGAGTTAAGCTGGGATTTTCCAAAATAATCCTGTTAACAGACTTGAAAGCACTTATCAGTTCTGTCTAATGAAGACATTAGAACACCATAACCTTTCCGG

CCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGCCTATGATCCCAGCGCTTTGGGAAACCAAGGTGGGTGGATCACCTGAGGTCAGGAGTTTGAGACCAGCTTGGCCAATAAGGTGAAACCCCATCTCTACAAAAATTAGTTGGGCGTGGTGGCGGCACCTGTAAATCCAGCTACTCAGGAGGCTGAGGCAAGAGGATTGCTTGAATCTGGGAGCCGGAGGTTGAAGTGAACTGAGATTGGGCCACTGCATTCTAGCCTGGGCGACAAGAGTGAAGCTCCATCTCAAAAAAAAAAAAAAAGATGAGGTTTTCCTTAAGAGCACTAACCTAGTATACTGCACAGGTGCCTGTATTCATGCATCCCACACAGAAAGAGAAAATACTTGTCTGAACTTGTCCATAAATTCAGAATCCTGCCCCTTAAC

Forward Primer: 5’-AGAACACCATAACCTTTCCGGCCC-3’



Reverse Primer Selection

Reverse Primer: 5’-GCGTGAGCCACTGCACCCGACTGG-3’

AGAACACCATAACCTTTCCGGCCCATTTTCTTTGTCAATAAGCGTTCTTGCCCTGTCAGCAGCTCACCTCCAGCTTTAGTTTTCXCATGACAGTAAGTCTATTACCCTCCTGATCTGTCTTCTGGCTCCTCCTACCCAGGATGGGGAAGGTTTTTGACTTTACTGATATTCTCAGAACAAATTTTGGGAAGTAAATATAAGGTTTTCCAGTCGGGTGCAGTGGCTCACGC

Amplified Fragment Will Be 230bp



5’-AGAACACCATAACCTTTCCGG

CCC-3’


Before ordering: Imagine the primers annealing to the DNA


AGCCACTGCACCCGACTGG-3’ Reverse Primer: 5’-GCGTG

Order From IDT



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

Use thin-wallPCR tubes

Use thin-wall tubes designed for PCR

Old School “Perkin Elmer 2400” PCR Thermocycler

Hot bonnet prevents condensation.

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.

Runs on antifreeze and refrigerant

Paper towels to adsorb leakingorange-colored antifreeze.

Antifreeze

R134a frigerant

Radiator &fan

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.

A typical PCR thermocycling program

55°C

62°C61°C

60°C59°C58°C57°C56°C

Gradient thermocyclers allow for optimization of the annealing temperature

What is RT-PCR?

Reverse Transcription- Polymerase Chain Reaction

RT-PCR is like any other PCR exceptit uses cDNA as a template.

How do you make cDNA?

cDNA can be created from RNA using

RNA-dependent DNA polymerase

(reverse transcriptase)

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’-

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.

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

Introduction to PCR and analysis of gene expression using RT-PCR

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