Lecture 10 Polymerase Chain Reaction PCR Flashcards
What is PCR?
Polymerase chain reaction is a technique used to make multiple copies of a segment of DNA. PCR is highly specific and can be used to amplify or copy a specific DNA target from a mixture of DNA molecules
Discovery of PCR using Taq DNA pol
Devised and developed by Kary Mullis 1983. 1988 paper in Science magazine describing the process, 1993 Nobel prize in chem for invention of PCR joint with Michael Smith for establishment of oligonucleotide based site directed mutagenesis and it’s development for protein study.
Isolation of Taq DNA pol from thermophilic bacterium Thermus aquatics in 1976 was essential for development of PCR - it meant that molecular biologists had a thermostable enzyme capable of repeat PCR cycling without need to add fresh DNA pol after each cycle
Thermostable DNA pol
Taq Pol lacks proofreading function so is error prone. Ok for most PCR uses but not for cloning where insertion of random mutation is undesirable.
Now a range of thermostable DNA pol are available many with higher fidelity e.g. Pfu from Pyrococcus furiosis and Vent from Thermococcus litoralis.
Basic requirements for PCR
1) DNA sequence of target region must be known
2) template DNA for amplification
3) primers - short oligonucleotides (18-25nt in length) that will bind to target sequences and prime DNA synthesis
4) thermostable DNA pol which is not inactivated by heating to 95°c e.g. Taq DNA pol
5) dNTPs deoxynucleoside triphosphates for each base (ACGT)
6) thermal cycler - a machine that can be programmed to carry out heating and cooling of samples over a no. Of cycles
Selection of oligonucleotide primers
2 oligonucleotides flanking the target are required, often called forward and reverse primers. One for each strand, pointing towards each other, each providing a 3’OH that will prime DNA pol to start synthesis
Primer design tips
1) primers should be 18-25nt long
2) similar TM (50-65°C) TM = melting temp. So that they will anneal at same temp.
3) similar GC content (40-60%)
4) a GC clamp at 3’ end to aid polymerization initiation
5) minimal secondary structure
6) minimal repeats e.g. not ATATATATATAT etc.
7) minimal runs of a single base (ideally no more than 4)
Potential primer problems
Self complimentarily or complementarity to other primer could result in hairpins, intrastrand pairing (self) or interstrand pairing (between primers).
Primer dimers
Primer annealing results in overhangs that can prime DNA synthesis - such pairings can be seen in PCR and are known as primer dimers
PCR steps
PCR amplification of DNA depends on a repeating 3 step cycle: denaturation, annealing, extension.
Multiple iterations of these steps result in exponential multiplication of the target DNA duplex even if template was a single copy at outset
Repeating these 3 steps 30-40 times will generate billions of copies of target DNA
PCR step 1: denaturation - 90-95°C
High temp separates dsDNA into ssDNA allowing acces for thermostable DNA Pol and allowing primers to bind. The presoak is an extended denaturing time that ensures all template DNA is ss
PCR step 2: annealing 55-65°C
At this cooler temp oligonucleotide primers can bind to the region’s next to target genes to be amplified. The binding is crucial, as primers act as the starting point for synthesis of new strands that will become copies of target region.
Primers are at a higher conc. Than template target so they preferentially anneal to the template rather than the complementary template strands reannealing to each other
PCR step 3: extension 68-72°C
Optimum temp. For DNA pol to incorporate dNTPs and extend primers into full length copies of target region.
Primers supply ‘3OH group so polymerase can add nucleotides. Extension time depends on speed of DNA pol and length of target sequence.
(In normal DNA synth in living organism the primer would be RNA synthesised by DNA primase using template to make the primer copy)
Typical PCR composition
In microlitres:
10x PCR reaction buffer : 5
50mM MgCl2 : 2
10mM of each dNTP: 1
10 micrometres forward primer: 1
10 micrometres reverse primer: 1
Template DNA : 1
Taq DNA pol (5U/ microlitres) : 0.5
Sterile distilled water: 38.5
Total vol. 50 microlitres
10xPCR buffer contains 500mM potassium chloride, 100 mM Tris-HCL (pH 8.3 at room temp) and 0.01% (w/v) gelatin
Analysis of DNA product
To confirm that pcr has amplified a product of correct size a sample can be loaded onto an agarose gel. Size of PCR product is estimated by comparison to a DNA hyperladder (molecular mass marker) which contains DNA molecules of known size and is loaded alongside on gel.
DNA is separated by electrophoresis (neg charged phosphodiester backbone of DNA makes it migrate towards pos electrode) larger DNA molecules move more slowly than smaller ones.
DNA is visualised by staining with ethidium bromide (or other fluorescent dye) that fluoresces under UV light
Reverse transcriptase PCR (RT-PCR)
Reverse transcriptase PCR (RT-PCR)
Making complementary DNA (cDNA) copy of RNA by reverse transcription followed by PCR - used to determine quantities of mRNA or genome copies of RNA viruses e.g. HIV and COVID 19
