Polymerase Chain Polymerase Chain ReactionReaction

““Xeroxing” DNAXeroxing” DNA

Life is not fair!Life is not fair!

1983—Kary Mullis, a scientist working for 1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along the Cetus Corporation was driving along US Route 101 in northern California when US Route 101 in northern California when he came up with the idea for the he came up with the idea for the polymerase chain reactionpolymerase chain reaction

1985—the polymerase chain reaction was 1985—the polymerase chain reaction was introduced to the scientific community at a introduced to the scientific community at a conference in Octoberconference in October

Still not fair!Still not fair!

Cetus rewarded Kary Mullis with a Cetus rewarded Kary Mullis with a $10,000 bonus for his invention$10,000 bonus for his invention

Later, during a corporate reorganization, Later, during a corporate reorganization, Cetus sold the patent for the PCR process Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-to a pharmaceutical company Hoffmann-LaRoche for $300 millionLaRoche for $300 million

Again I say, life is not fair!Again I say, life is not fair!

Polymerase Chain ReactionPolymerase Chain Reaction

PCR for short!PCR for short!

A technique for making MANY copies of a A technique for making MANY copies of a particular DNA sequenceparticular DNA sequence

Allows us to start with VERY SMALL Allows us to start with VERY SMALL samplessamples

We need a large sample to perform We need a large sample to perform electrophoresis and other analyseselectrophoresis and other analyses

PCR: Amplification of DNAPCR: Amplification of DNA

PCR: Amplification of DNAPCR: Amplification of DNAOften, only a small amount of DNA is Often, only a small amount of DNA is availableavailable A drop of bloodA drop of blood A rare cell typeA rare cell type

Two methods currently exist for amplifying Two methods currently exist for amplifying the DNA or making copiesthe DNA or making copies Cloning—takes a long time for enough clones Cloning—takes a long time for enough clones

to reach maturityto reach maturity PCR—works on even a single molecule PCR—works on even a single molecule

quicklyquickly

PCR basis: Directionality of DNA PCR basis: Directionality of DNA backbonebackbone

Recall that DNA is antiparallelRecall that DNA is antiparallel

Heat causes denaturing—H-bonds Heat causes denaturing—H-bonds between base pairs “break”between base pairs “break”

AnnealingAnnealing

Fancy word for renaturingFancy word for renaturing When denatured strands of When denatured strands of

DNA cool, it can renatureDNA cool, it can renature HYDROGEN BONDS RE-HYDROGEN BONDS RE-

FORM!FORM! Complimentary base pairs Complimentary base pairs

must line up in order for must line up in order for this to happenthis to happen

The denaturing and The denaturing and annealing of DNA is an annealing of DNA is an important part of PCRimportant part of PCR

DNA Polymerase moves in a DNA Polymerase moves in a 5’5’3’ direction3’ direction

DNA polymerase is the key to “xeroxing” DNA polymerase is the key to “xeroxing” copies of the DNA sample!copies of the DNA sample!DNA polymerase is an enzyme that uses DNA polymerase is an enzyme that uses an existing an existing SINGLESINGLE strand of DNA as a strand of DNA as a template to form a new complementary template to form a new complementary strand.strand.To get started, DNA polymerase needs a To get started, DNA polymerase needs a small complementary sequence, called a small complementary sequence, called a primerprimer

Note: SYNTHESIS occurs in the Note: SYNTHESIS occurs in the 5’5’3’ direction3’ direction

Where do we get loads of this DNA Where do we get loads of this DNA polymerase enzyme?polymerase enzyme?

Most commonly, our good buddy Most commonly, our good buddy E. coliE. coli

Trouble is, Trouble is, E. coliE. coli’s DNA polymerase is ALSO ’s DNA polymerase is ALSO denatured at the high temperatures needed to denatured at the high temperatures needed to denature the DNA!denature the DNA!

This problem was solved by looking to bacteria This problem was solved by looking to bacteria that live in hot springs such as Old Faithfulthat live in hot springs such as Old Faithful

The DNA polymerase of these bacterial cells can The DNA polymerase of these bacterial cells can withstand the high temperatures needed withstand the high temperatures needed WITHOUT having the enzyme denature!WITHOUT having the enzyme denature!

Old Faithful!Old Faithful!

Taq polymeraseTaq polymeraseThis is the name of the most common This is the name of the most common heat-resistant polymerase extracted from heat-resistant polymerase extracted from these thermophilic bacteria.these thermophilic bacteria.

Thermus aquaticusThermus aquaticus is the genus and is the genus and species name of the bacteria, Taq for species name of the bacteria, Taq for short!short!

Making PCR twice as fast!Making PCR twice as fast!The stretch of DNA to be amplified is often The stretch of DNA to be amplified is often referred to as the “target sequence”referred to as the “target sequence”The template DNA is usually a larger stretch of The template DNA is usually a larger stretch of sample DNA that contains the target sequencesample DNA that contains the target sequenceThe The primersprimers are short stretches of DNA that are short stretches of DNA that flank the target sequence and are flank the target sequence and are complementary to opposite strands of the longer complementary to opposite strands of the longer sample DNAsample DNAThe primer attaches to the beginning of the The primer attaches to the beginning of the target sequence on one strand and the END of target sequence on one strand and the END of the target sequence on the other strand of DNAthe target sequence on the other strand of DNABig deal?Big deal?You bet! Now both strands of the sample DNA You bet! Now both strands of the sample DNA are templates for the polymerase enzymes!!are templates for the polymerase enzymes!!

PCR: Essential ReagentsPCR: Essential Reagents

An excess of spare nucleotidesAn excess of spare nucleotides

An excess of primersAn excess of primers We want the primers to bind to the sample We want the primers to bind to the sample

DNA once it is denatured to prevent the DNA once it is denatured to prevent the strands from reannealingstrands from reannealing

PLENTY of DNA polymerase [Taq PLENTY of DNA polymerase [Taq polymerase in particular]polymerase in particular]

Sample template of DNASample template of DNA

PCR: Pipetting the reagents into PCR: Pipetting the reagents into the reaction tubethe reaction tube

““PCR machine”: The thermocyclerPCR machine”: The thermocycler

A programmable machine A programmable machine that can change that can change temperatures with great temperatures with great accuracy and at precise accuracy and at precise times—it’s like a fancy times—it’s like a fancy incubatorincubator

9494°C is always the first °C is always the first temperature set in the temperature set in the programprogram

DNA denatures at this DNA denatures at this temperaturetemperature

First denaturationFirst denaturation

As the temperature is raised to 94As the temperature is raised to 94°C, the °C, the target DNA denaturestarget DNA denatures

At these high temperatures, there will be At these high temperatures, there will be NO BINDING of any sequencesNO BINDING of any sequences

The reaction mixture is left at 94°C for 5 The reaction mixture is left at 94°C for 5 minutes to allow the DNA to completely minutes to allow the DNA to completely denaturedenature

Binding of primers during annealingBinding of primers during annealing

As the tube is As the tube is cooled, the cooled, the primers will primers will bind to the bind to the sequences sequences that flank the that flank the target target sequence on sequence on the two the two strandsstrands

Thermocycler lowered to annealing Thermocycler lowered to annealing temperaturetemperature

The tube is The tube is lowered to lowered to 5050°C for 2 °C for 2 minutes to minutes to allow the allow the primers to primers to anneal to the anneal to the template template DNA.DNA.

PCR: Polymerization by High –PCR: Polymerization by High –temperature DNA polymerasetemperature DNA polymerase

After the primers have bound to the After the primers have bound to the flanking sequences, the DNA polymerase flanking sequences, the DNA polymerase will start to synthesize the complementary will start to synthesize the complementary strand. strand.

The end result will be four copies of the The end result will be four copies of the target sequence, doubling the two present target sequence, doubling the two present at the start.at the start.

Thermocycler at polymerization Thermocycler at polymerization temperaturetemperature

The temperature The temperature of the reaction of the reaction mixture is raised mixture is raised to 72to 72°C for 3 °C for 3 minutes to allow minutes to allow the polymerase to the polymerase to copy the target copy the target sequence. The sequence. The temperature is temperature is raised to 94°C raised to 94°C and the cycle is and the cycle is repeatedrepeated

PCR denaturation step repeatedPCR denaturation step repeated

When the When the temperature is temperature is again raised to again raised to 9494°C, both the °C, both the original original template DNA template DNA and the copies and the copies from the last from the last cycle denaturecycle denature

PCR: Annealing and PCR: Annealing and polymerization repeatedpolymerization repeated

Now, both the two original target Now, both the two original target sequences AND the two copies of the sequences AND the two copies of the sequence can act as templates. sequence can act as templates.

As the reaction is cooled, primers bind to As the reaction is cooled, primers bind to the templates, and the polymerase makes the templates, and the polymerase makes copies in the opposite directions. copies in the opposite directions.

You can see in this next image why You can see in this next image why primers are needed in both directions.primers are needed in both directions.

Denaturation repeated a third timeDenaturation repeated a third time

The temperature is raised yet again, and The temperature is raised yet again, and the double strands fall apartthe double strands fall apart

There are now EIGHT template strands There are now EIGHT template strands available for making copiesavailable for making copies

Our hero! The thermocycler!Our hero! The thermocycler!

The developers of PCR and the early The developers of PCR and the early researchers who used the technique spent long researchers who used the technique spent long hours transferring tubes between the different hours transferring tubes between the different hot water baths. hot water baths. Fortunately, this tedious process is almost Fortunately, this tedious process is almost entirely automated by the thermocycler. The entirely automated by the thermocycler. The times and temperatures described in this tutorial times and temperatures described in this tutorial for each step are averages, and different for each step are averages, and different experiments call for different conditionsexperiments call for different conditionsThe machine can be programmed for any The machine can be programmed for any changes in temperature the experimenter finds changes in temperature the experimenter finds useful.useful.

Repeat, repeat, repeat!Repeat, repeat, repeat!

After this denaturing and synthesizing After this denaturing and synthesizing process is repeated for 30 cycles, the process is repeated for 30 cycles, the sequence will have been amplified a sequence will have been amplified a BILLION timesBILLION times

This geometric increase in the amount of This geometric increase in the amount of target sequence can be achieved in just a target sequence can be achieved in just a few hours rather than the much longer few hours rather than the much longer times required by older methods!times required by older methods!

After 30 cycles this becomes

one billion! 230

What next?What next?

Now that the most minute sample of DNA Now that the most minute sample of DNA has been amplified, there is a large has been amplified, there is a large enough PCR product to be enough PCR product to be electrophoresed.electrophoresed.

Applications of PCRApplications of PCR

The first application dealt with detection of The first application dealt with detection of genetic mutationsgenetic mutations

Now the smallest trace evidence from Now the smallest trace evidence from crime scenes can be amplified in order to crime scenes can be amplified in order to provide DNA fingerprinting dataprovide DNA fingerprinting data