PCR

Pallavi Angule
LCBT - 13

5/19/2011

What is Polymerase Chain Reaction PCR ?

Polymerase chain reaction (PCR) is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA , generating thousands to millions of copies of a particular DNA sequence.

In 1968 Nobel laureate H. Gobind Khorana, and allows the amplification of specific DNA sequences. Discovered in 1983 by Kary Mullis 1993 Nobel Prize in Chemistry Dr. Kary Mulli

The Japan Prize in the same year

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Practical uses of PCR

Disease detection Cloning Forensics Food quality control Paternity testing Identification

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Computer And PCR Analogies Computer PCR

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Hardware
Feedback loop Clock speed Operating system CPU Coprocessor

o Thermal cyclar & plate reader o Cycles o Cycles times o Buffer o DNA polymerase activity o 3 nuclease activity (proofreading activity) 5 nuclease activity (Detection strategies)
o Optical fibers o Targets(Genes) o Primer pairs

o o o

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Bus Applications Search algorithms(drivers) Input Output Multitasking

o dNTPs o Amplicons (Signals) o Multiplexing(Parallel processing)

Referance: PCR Applications protocols for functional genomics By Michael A. Innis, John J. Sninsky, David H. Gelfand
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RT-PCR : an RNA strand is first reverse transcribed into its DNA complement (complementary DNA, or cDNA) using the enzyme reverse transcriptase, and the resulting cDNA is amplified using traditional PCR.

How it works..????? RT_PCR

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PCR Reaction Components

DNA template:
Amount of DNA present
Less DNA means more cycles

Purity
Interfering factors, eg. enzymes, salts

Degradation
PCR more forgiving of degraded DNA

Contamination
Amplification products

Presence of poisons
Eg. EDTA which scavenges Mg++

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PCR Reaction Components

Primers:
Age Number of freeze-thaws Contamination Amount
Can vary over a wide range (50X) 100-500 nM typical Too low: low amplification Too high: low amplification
http://info.med.yale.edu/genetics/ward/tavi/p05.html

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PCR Reaction Components

Nucleotides(dNTPs):
20-400 uM works well
Too much: can lead to mispriming and errors Too much: can scavenge Mg++ Too low: faint products

Age Number of freeze-thaws

Just 3-5 cycles is enough to make PCRs not work well

Dilute in buffer (eg. 10mM Tris pH 8.0 to prevent acid hydrolysis) Contamination

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http://info.med.yale.edu/genetics/ward/tavi/p13.html

PCR Reaction Components

Mg++ ions: Mg is an essential cofactor of DNA polymerase Amount can vary
0.5 to 3.5 uM suggested Too low: Taq wont work Too high: mispriming

http://info.med.yale.edu/genetics/ward/tavi/p14.html 19 May 2011 9

PCR Reaction Components

DNA Polymerase: Thermostable?
Activity declines with time at 95C

Matches buffer? Age Contamination Concentration: Typically 0.5 to 1.0 U/rxn

http://info.med.yale.edu/genetics/ward/tavi/p12.html 10

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PCR Reaction Components

Bottom Line:
All components work over a wide range. Need to avoid contamination. Optimization by trial-and-error. Good experimental planning. Able to troubleshoot.

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PCR Cycling Parameters

Denaturation Temp Annealing Temp Extension Temp Time Number of Cycles Reaction Volume Odd Protocols

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PCR Cycling Parameters

Denaturation Step: Must balance DNA denaturation with Taq damage 95C for 30 - 60s typically is enough to denature DNA Even 92C for 1s can be enough Taq loses activity at high temps:
Half-life at 95C: 40 min Half-life at 97.5C: 5 min

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http://info.med.yale.edu/genetics/ward/tavi/p08.html

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PCR Cycling Parameters

Annealing Step: Most critical step
Calculate based on Tm
Often does not give expected results

Trial-and-Error
Almost always must be done anyway Too hot: no products Too cool: non-specific products

Gradient thermocyclers very useful Typically only 20s needed for primers to anneal
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PCR Cycling Parameters

Extension Step: Temperature typically 72C
Reaction will also work well at 65C or other temps

Time (in minutes) roughly equal to size of the largest product in kb

Polymerase runs at 60bp/s under optimum conditions

Final long extension step mostly unnecessary

http://info.med.yale.edu/genetics/ward/tavi/p08.html 19 May 2011 http://info.med.yale.edu/genetics/ward/tavi/p10.html 15

PCR Cycling Parameters

Number of Cycles:
Number of source molecules:
>100,000: 25-30 >10,000: 30-35 >1,000: 35-40 <50: 20-30 fb. nested PCR

Do not run more than 40

Virtually no gain Extremely high chance of nonspecific products

Best optimized by trial-and-error

http://info.med.yale.edu/genetics/ward/tavi/p08.html 19 May 2011 16

PCR Cycling Parameters

Reaction Volume: Doesnt affect PCR results as long as volume is within limits. Heated lid important. 5ul, 20ul, 100ul all work. Slightly higher yield with lower volumes.

http://info.med.yale.edu/genetics/ward/tavi/p03.html 19 May 2011 17

PCR Cycling Parameters

Odd Protocols: Hot-Start PCR
Taq is added last

Touchdown PCR
Annealing temp is progressively reduced

Gradient PCR
Heterologous primers

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Basic Experimental Design

A well-designed experiment can keep you from ever getting into trouble! A poorly-designed experiment is asking for problems!!!!

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Basic Experimental Design

Main point: Always use CONTROLS Positive control
So youll know what a successful result looks like.

Negative control
Lets you know if you have contamination.

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Experimental Design: Controls

No positive or negative controls What does this result mean??

Only a positive control How do we know the result isnt due to contamination?

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Both positive and negative controls Results can be interpreted with confidence.
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Experimental Design: Replication

Our unknown is definitely positive... but how sure are we?

We ran the same sample three times. Is our unknown really positive?

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References :

PCR troubleshooting by Dave Palmer, Bio-Rad PCR applications: protocols for functional genomics Michael A. Innis, John J. Sninsky, David H. Gelfand 0 Reviews Academic Press, Year 1999 , page no. 25 -29 http://info.med.yale.edu/genetics/ward/tavi/p08.html http://info.med.yale.edu/genetics/ward/tavi/p10.html

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Thank You

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