1983—Kary Mullis, a scientist working for the Cetus Corporation was driving along US Route 101 in northern California when he came up with the idea for the polymerase chain reaction
1985—the polymerase chain reaction was introduced to the scientific community at a conference in October
Still not fair!
Cetus rewarded Kary Mullis with a $10,000 bonus for his invention
Later, during a corporate reorganization, Cetus sold the patent for the PCR process to a pharmaceutical company Hoffmann-LaRoche for $300 million
Again I say, life is not fair!
Polymerase Chain Reaction
PCR for short!
A technique for making MANY copies of a particular DNA sequence
Allows us to start with VERY SMALL samples
We need a large sample to perform electrophoresis and other analyses
PCR: Amplification of DNA
PCR: Amplification of DNA
Often, only a small amount of DNA is available
A drop of blood
A rare cell type
Two methods currently exist for amplifying the DNA or making copies
Cloning—takes a long time for enough clones to reach maturity
PCR—works on even a single molecule quickly
PCR basis: Directionality of DNA backbone
Recall that DNA is antiparallel
Heat causes denaturing—H-bonds between base pairs “break”
Fancy word for renaturing
When denatured strands of DNA cool, it can renature
HYDROGEN BONDS RE-FORM!
Complimentary base pairs must line up in order for this to happen
The denaturing and annealing of DNA is an important part of PCR
DNA Polymerase moves in a 5’ 3’ direction
DNA polymerase is the key to “xeroxing” copies of the DNA sample!
DNA polymerase is an enzyme that uses an existing SINGLE strand of DNA as a template to form a new complementary strand.
To get started, DNA polymerase needs a small complementary sequence, called a primer
Note: SYNTHESIS occurs in the 5’ 3’ direction
Where do we get loads of this DNA polymerase enzyme?
Most commonly, our good buddy E. coli
Trouble is, E. coli ’s DNA polymerase is ALSO denatured at the high temperatures needed to denature the DNA!
This problem was solved by looking to bacteria that live in hot springs such as Old Faithful
The DNA polymerase of these bacterial cells can withstand the high temperatures needed WITHOUT having the enzyme denature!
This is the name of the most common heat-resistant polymerase extracted from these thermophilic bacteria.
Thermus aquaticus is the genus and species name of the bacteria, Taq for short!
Making PCR twice as fast!
The stretch of DNA to be amplified is often referred to as the “target sequence”
The template DNA is usually a larger stretch of sample DNA that contains the target sequence
The primers are short stretches of DNA that flank the target sequence and are complementary to opposite strands of the longer sample DNA
The primer attaches to the beginning of the target sequence on one strand and the END of the target sequence on the other strand of DNA
You bet! Now both strands of the sample DNA are templates for the polymerase enzymes!!
PCR: Essential Reagents
An excess of spare nucleotides
An excess of primers
We want the primers to bind to the sample DNA once it is denatured to prevent the strands from reannealing
PLENTY of DNA polymerase [Taq polymerase in particular]
Sample template of DNA
PCR: Pipetting the reagents into the reaction tube
“ PCR machine”: The thermocycler
A programmable machine that can change temperatures with great accuracy and at precise times—it’s like a fancy incubator
94 °C is always the first temperature set in the program
DNA denatures at this temperature
As the temperature is raised to 94 °C, the target DNA denatures
At these high temperatures, there will be NO BINDING of any sequences
The reaction mixture is left at 94°C for 5 minutes to allow the DNA to completely denature
Binding of primers during annealing
As the tube is cooled, the primers will bind to the sequences that flank the target sequence on the two strands
Thermocycler lowered to annealing temperature
The tube is lowered to 50 °C for 2 minutes to allow the primers to anneal to the template DNA.
PCR: Polymerization by High –temperature DNA polymerase
After the primers have bound to the flanking sequences, the DNA polymerase will start to synthesize the complementary strand.
The end result will be four copies of the target sequence, doubling the two present at the start.
Thermocycler at polymerization temperature
The temperature of the reaction mixture is raised to 72 °C for 3 minutes to allow the polymerase to copy the target sequence. The temperature is raised to 94°C and the cycle is repeated
PCR denaturation step repeated
When the temperature is again raised to 94 °C, both the original template DNA and the copies from the last cycle denature
PCR: Annealing and polymerization repeated
Now, both the two original target sequences AND the two copies of the sequence can act as templates.
As the reaction is cooled, primers bind to the templates, and the polymerase makes copies in the opposite directions.
You can see in this next image why primers are needed in both directions.
Denaturation repeated a third time
The temperature is raised yet again, and the double strands fall apart
There are now EIGHT template strands available for making copies
Our hero! The thermocycler!
The developers of PCR and the early researchers who used the technique spent long hours transferring tubes between the different hot water baths.
Fortunately, this tedious process is almost entirely automated by the thermocycler. The times and temperatures described in this tutorial for each step are averages, and different experiments call for different conditions
The machine can be programmed for any changes in temperature the experimenter finds useful.
Repeat, repeat, repeat!
After this denaturing and synthesizing process is repeated for 30 cycles, the sequence will have been amplified a BILLION times
This geometric increase in the amount of target sequence can be achieved in just a few hours rather than the much longer times required by older methods!
After 30 cycles this becomes one billion! 2 30
Now that the most minute sample of DNA has been amplified, there is a large enough PCR product to be electrophoresed.
Applications of PCR
The first application dealt with detection of genetic mutations
Now the smallest trace evidence from crime scenes can be amplified in order to provide DNA fingerprinting data