We live in a time where genetics are helping us understand more and more of the world around us – from treating disease more precisely to untangling revolutionary historyIn order to achieve this, we need to find and examine specific pieces of DNA. One of the fundamental methods for doing so is Polymerase Chain Reaction (PCR).
PCR is an invaluable laboratory technique that detects genetic material from a specific organism and uses repeated temperature cycles to make millions of copies of a specific region of DNA.
Scientists have developed many different types of PCR. Some of the most used methods in life science laboratories are conventional PCR, quantitative real-time PCR (qPCR), and reversed transcriptase PCR (RT-PCR).
The techniques are widely utilized for anything from quantification of pathogens and diagnostics tests, such as COVID-19 testing, to cancer detection and detecting infectious agents in food. Other uses for PCR include genetic fingerprinting, detection of mutation, cloning genes and PCR sequencing.
Conventional PCR (cPCR)
Conventional PCR is the most basic type of PCR reaction. It gives qualitative results and requires a post-PCR step for detection or visualization of the DNA. Often, conventional PCR reactions are loaded onto an agarose gel and are resolved by size via electrophoresis. The DNA is then visualized by using an intercalating dye and a UV light source.
A major advantage to conventional PCR is the low cost and that most research facilities already have access to conventional thermocyclers.
Limitations of this type of end-point PCR include low sensitivity and non-quantitative results. In addition, the extra step for detection of DNA makes the process slower than e.g. real-time PCR.
Quantitative Real-time PCR (qPCR)
In Quantitative Real-Time PCR (qPCR), DNA molecules are tagged with fluorescent dye which is used to monitor and quantify PCR products in real-time. In other words, it measures the amplification as it occurs.
It is faster than conventional PCR as there is no post-PCR step for DNA detection, which also reduces the risk of cross-contamination. In addition, it can determine the number of target copies in the original specimen, unlike conventional PCR.
Reverse Transcriptase PCR (RT-PCR)
Another common PCR type is Reverse Transcriptase PCR (RT-PCR), which is used when the starting material is RNA.
This method also monitors the reaction in real-time. It amplifies target RNA by transcribing the template RNA and forming complementary DNA. The new complementary DNA can then be amplified via conventional PCR or qPCR.
Liquid handling in PCR protocols
Liquid handling is a big part of carrying out different types of PCR in a laboratory. Setting up a PCR assay requires multiple steps of transferring liquid between many reaction vessels. This can be done either manually or through an automation solution.
It is of utmost importance that the liquid is handled correctly to achieve great accuracy and avoid cross-contamination. But a handheld pipetting routine is a tedious and time-consuming task that may cause strain-injuries and back pain. Therefore, many laboratories implement automation solutions to unburden their staff, avoid human errors and increase throughput.
Our contribution to different types of PCR workflows is the pipetting robot, flowbot® ONE, that allows you to easily automate the liquid handling and set up applications such as PCR assays.
Book your free demo today to see what Flowbot® ONE can do for you.
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