What is PCR?
-Polymerase Chain Reaction
-Amplification of DNA (exact copies)
-Based on DNA polymerase being able to synthesise a new strand of DNA complementary to the original ‘template’ strand.
-Starts with a single stranded piece of DNA.
-Uses Taq polymerase for repeated cycles.
-With each cycle there is an exponential increase in strands
-Essentially copies aspect of replication.
What is the prokaryotic model of PCR?
- DNA helicase separates dsDNA strands, binds to structure and energetically breaks the H bonds between them. Phosphodiester bonds do not break.
- Primases attach RNA primers to the open strands.
- DNA polymerase binds to double stranded structure and slides along leading strand of DNA, producing complimentary strand, from 5’ to 3’.
- Lagging strand, produce smaller fragments of DNA (Okazaki fragments) =pointless so ligase joins (ligates) these together.
What does alpha polymerase do?
Starts initial process on leading strand.
What does epsilon polymerase do?
Produces lagging strand
What does delta polymerase do?
Takes over to produce DNA product.
What is needed from replication process for PCR?
-Leading strand
-Template DNA
-Polymerases
Why use PCR?
- Sensitive – amplify as little as one molecule of DNA.
- Specific – can amplify a unique target sequence, stringency depends on temperature and Mg2+.
- Cheap
- Rapid – results available in a few hours.
- Robust – DNA is very stable and can be amplified from old and degraded samples.
What goes into PCR tube?
-Template – double stranded DNA, the sequence to be amplified
-2 primers – to prime synthesis in both directions (small ssDNA molecules, 6-30 bases, chemically synthesised)
-Polymerase – copies the template, extending from 3’ end of primer.
-dNTPS – deoxyribonucleotide triphosphates, dATP, dCTP, dGTP, dTTP
-Magnesium – essential for polymerase.
-Buffer – maintains optimal pH
-Water - makes up final conc
Nucleotide characteristics
-Building blocks
-Phosphate (strength), sugar (orientation), base (specificity)
-Thymine, Cytosine, Adenine, Guanine
Taq Polymerase characteristics
-All living cells contain polymerase.
-Copy DNA accurately
-Enzyme has 3 regions:
1. Synthesis
2. Proof reading
3. Primer removal
-Taq polymerase can withstand high temperatures.
Primers characteristics
-2 primers – one for each strand
-Single stranded DNA (oligonucleotide)
-Length of 18-24 bases:
* Too long – hybridise too slowly.
* Too short – won’t be specific, may bind elsewhere.
-40-60% G/C content
-Start and end with 1-2 G/C pairs.
-Melting temp of 50-60C.
-Primer pairs should have temp within 5C of each other.
-3’ end must be complementary to the template DNA
-Primer pairs should not have complementary regions.
Magnesium characteristics
-MgCl2
-A co-factor
-A non-protein component of the reaction that’s needed to enable the activity of the catalysis. Maybe metallic or compound based.
-Magnesium acts to enhance the enzymatic activity (specifically of DNA polymerase) thereby supporting DNA application.
Buffer characteristics
-10x buffer
-Optimal pH is 8-9.5
-Tris HCl
-Potassium ions (KCl) – promotes annealing.
*May be replaced by ammonium sulphate, which destabilises base pairing bonds.
Describe PCR reaction
-Denaturation – break bonds by heating to 90C
-Annealing – primer binds to specific sites, temp reduced to 55C.
-Elongation – heat to 72C, optimal temp for Taq polymerase to operate.
1st cycle – synthesis of a strand of DNA in a test tube
2nd cycle – synthesis of 2 strands in a test tube
The rest – simultaneous synthesis of both strands
-30 cycles = >1 billion copies
Function of sliding clamp
Holds the polymerase on the ssDNA.
What makes a good PCR?
- Clean gloves, lab coat, pipettes, and benches
- Dedicated areas in lab
- Care with tubes and templates
- Don’t bring in old samples.
- Use UV to ensure equipment isn’t contaminated.
- Take your time, pipette accurately.
- Pipette negative controls and samples first before positive controls.
Uses of PCR
- Able to amplify and manipulate DNA.
- Good when DNA is scarce.
- Diagnosis of genetic diseases.
- Detect genetically modified material.
- Ancient DNA
- Forensic analysis of DNA samples
- Manipulate DNA – create artificial fragments and then introduce into host cells and gets incorporated into genome (genetic modification)
- Knock out genes – study gene function.
- Fuse host proteins with GFP
Differences between end point and real time PCR
End point:
- Cheap
- Semi quantitative at best – band intensity
- Sequencing, genotyping, cloning
- See results at end, plateau
Real time:
- More expensive
- Quantity of PCR is proportional to amount of template
- Quantification of gene expression, microarray verification, quality control and assay validation, SNP genotyping, copy number variation, viral quantification, siRNA /RNAi experiments
- Measures at exponential phase – more precise
What are reference genes?
- ‘Housekeeping genes’
- Constant level of expression – not affected by experimental factors.
- Essential to support validity of qPCR results.
- Confirms RNA extraction was good and efficient.
- Supports conclusions of expression levels (credibility)
What is the source of RNA in reverse transcriptase PCR?
- Gene expression (mRNA): diseased versus healthy/drug effects/environment changes
- RNA virus infection levels
Briefly describe reverse transcriptase PCR
- Convert RNA (often mRNA) to cDNA. Use reverse transcriptase (PCR must start with DNA).
- Amplify DNA by PCR (including qPCR)
Theory of qPCR
In the reaction:
- Thermos lightcycler - detects fluorescence
- Fluorescent - SYBR green or TaqMan
- Mastermix.
- PCR product is measured as it is produced e.g., by incorporating fluorescent marker into the product.
- The cycle number at which the fluorescence reaches a threshold value is measured.
- The lower the Ct value, the greater the quantity of DNA/cDNA in the starting template
Why is PCR clinically valuable?
- Sensitive – can amplify as little as one molecule of DNA.
- Specific – can amplify a unique target sequence stringency depend on temperature and Mg2+.
- Cheap – relatively
- Rapid – results available in a few hours.
- Robust – DNA is very stable, can be amplified from old and degraded samples.
Purpose of genotyping the patient
- Diagnosis of genetic traits
- Detection of carriers of genetic traits
- Tissue matching (HLA typing)
- Predicting response to drugs (pharmacogenetics)
