One might ask, what is PCR and what does it stand for? PCR stands for Polymerase chain reaction. Polymerase chain reaction (PCR) is a technique used in molecular biology to amplify a single copy or a few copies of a piece of DNA. There are many different forms of PCR reactions, such as Real Time PCR (rt PCR)/quantitative PCR (qPCR) and also multiplex PCR, nested PCRs or digital PCR and in silico PCR. The basics of PCR and qPCR are explained here, together with some tips and tricks. For a great PCR reaction, it is key to use qualitative components. GC biotech supplies Bioline products, which are produced under a strict quality regime and assure a high quality for an affordable price.
The PCR protocol consists of a series of repeated temperature changes, called cycles, with each cycle commonly consisting of 2–3 discrete temperature steps. These steps of PCR are as follows:
Denaturing – when the double-stranded template DNA is heated to separate it into two single strands.
Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA.
Extending – when the temperature is raised and the new strand of DNA is made by the polymerase enzyme.
These three PCR steps are repeated 20-40 times, doubling the number of DNA copies each time.
Performing a good Polymerase chain reaction requires an optimized reaction buffer and a high-quality, thermostable DNA polymerase. The enzyme polymerase ‘paste’ the single oligonucleotides on the separated DNA fragment of interest in every annealing step of the PCR cycle.
Polymerases is an enzyme isolated in thermophilic bacterium, to be stable at higher temperatures than normal, so it would not denatured by the repeated heat treatments in the PCR cycles resulting in four different polymerases types. Standard DNA polymerases, Hot-start polymerases, high-fidelity polymerases and polymerases for amplification of long amplicons.
Standard DNA polymerases, like MyTaq are suitable for PCRs detecting of amplified product and estimation of product size
Hot-start polymerases are suitable for suppressing of nonspecific product amplification for setup and to increase yield of the product of interest like the MyTaq HS performs.
High-fidelity polymerases remove erroneous bases incorporated in the new DNA strand or increase the accuracy of the synthesis for the template DNA as the results show on the VELOCITY enzyme.
Polymerases for amplification of long amplicons is a combination of the accuracy of a proofreading polymerase and standard DNA polymerases as RANGER.
What is qPCR?
Real Time PCR or quantitative PCR combines PCR amplification and detection in one single application. There a two main types of qPCR reactions.
The first one utilizes fluorescent dyes like SYBR, which intercalate with the double stranded DNA during thermal cycling. This enables the qPCR cycler to detect of the amount of product by measuring the amount of fluorescence.
On the other hand, there are the qPCR probe mixes like with use probes like Taqman or Scorpion. These probes are designed to hybridize onto the PCR product, which mostly is the gene of interest. The probe contains a fluorescent and a quencher. While both are attached to the probe, the quencher suppresses fluorescence, but when the product of interest is amplified via qPCR the polymerase removes the probe by destroying it, thus detaching probe and quencher which results in a fluorescent signal.
Using qPCR mixes like Sensifast Probe mix, a mix created by Bioline: the PCR Company, you can set up a multiplex qPCR reaction by attaching different fluorescent colors to the probes for different genes or PCR products.
RT PCR (not to be confused with reverse transcriptase PCR), can be performed on both DNA and cDNA, where the second is derived from RNA using a technique called cDNA synthesis. This method, which in nature is performed by retro viruses, uses a reverse transcriptase, to reverse engineer DNA from mRNA and is also known as reverse transcriptase PCR. As the amount of RNA is directly related to the amount of protein produced and gene activity this method is perfect for identifying under- or overexpression of genes under different conditions.
qPCR eliminates the need to detect products using gel electrophoresis, and more importantly the method is truly quantitative.
General considerations and optimizations
- RNase-AWAY the bench and pipettes
- Use a multichannel pipette
- Always use filter tips
- Separate setup and PCR areas
- Ideally a room for extraction, one for setup and a third for the real-time PCR, if this is not possible or practical use hoods with a UV lamp, particularly for the setup.
- Use dUTP (and UNGase)
- Generally we recommend not using dUTP, because it is only effective if all the researchers do so. Using dUTP has also been shown to be inefficient as it increases the Ct values by reducing reaction efficiency.
Optimization PCR protocol
- PCR primer and/or probe design may not be optimal
- Samples may contain PCR inhibitors
- Inaccurate sample and reagent pipetting
- Standard curve may not have been properly analyzed
Optimization qPCR protocol
- Increase annealing/extension temperature (up to a maximum of 65 °C)
- Adjust annealing/extension time, recommended 15 s (and up to a maximum of 30 s)
- If necessary, redesign primers and or probes using primer design software
- As a last resort you can increase MgCl2 concentration from 3 mM up to a maximum of 6 mM
An early Ct is not indicative of sensitivity, a dilution series should be run to establish the lower limits of detection. Running a dilution series will also show qPCR efficiency (the slope will show if the product is doubling each time) and it will indicate inhibitors, as the effect of inhibitors in the sample will be reduced during dilution.
Controls are essential and include:
- NTC (No Template Control) – checks for primer dimers or template contamination of reaction components
- No RT (No Reverse Transcriptase Control) – checks for genomic DNA contamination when working with RNA
- NAC (No Amplification Control) – will show you degrading probe in TaqMan assays
- Positive control – checks that reagents and primers work, especially importance if trying to show absence of expression of a gene