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PCR in Laminar Flow Hoods

Polymerase chain reaction (PCR), sometimes referred to as ‘molecular photocopying’, is a cost-effective method widely used to make copies of DNA in vitro (in a test tube, not a living organism). This method is quick and produces the large amounts of DNA required for genetic testing. PCR was created by Kary B. Mullis in 1983, and he later received a Nobel Prize in Chemistry for his work. Since then, PCR has become a crucial technique in genetic testing, genetics research, and identifying infectious agents like viruses.

Applications of PCR include rare mutation and viral load detection, copy number variation, gene expression analysis and NGS library quantification
Applications of PCR

What is PCR?

PCR is a process used to replicate DNA. It relies on a molecule called DNA polymerase, which can create a new DNA strand that matches a template strand.

DNA polymerase belongs to a family of enzymes responsible for synthesis of DNA molecules, which are essential for the replication of DNA. The DNA polymerase used for PCR is called Taq polymerase. The enzyme isolated from a heat-tolerant bacterium called Thermus aquaticus. To produce new DNA, the Taq polymerase is given a primer. A primer is a short sequence of nucleotides that provide a starting point for DNA synthesis.

During the process, a short, specific DNA sequence is multiplied many times. PCR uses three temperature-dependent steps:

  1. The DNA sequence is heated, separating it into single strands.
  2. The temperature is reduced to enable the single DNA strands to attach to primers.
  3. The temperature is increased again to make a heat-resistant version of the DNA polymerase. From this, two new DNA strands are created.

This whole process is repeated 30-40 times. Each time you get billions of copies of the specific DNA sequence. Once the samples are prepared, the entire process can be automated and completed in just a few hours.

Stages of PCR amplification
Stages of PCR amplification

Contamination During PCR

It is important to prevent contamination during PCR as this can easily affect the process. Preventing contamination is vital if you want reliable and accurate results. Contamination can occur in three main ways:

  • Between different samples
  • Between different DNA molecules
  • From previously amplified DNA, known as amplicon carryover

The most common source of contamination is amplicon carryover. This happens when a tube is opened, and previously amplified DNA is released into the air. Amplicon carryover can contaminate equipment, surfaces, and even the ventilation system. Other sources of contamination include contamination from other disposable materials and chemicals.

If DNA fragments from the lab environment get into your PCR reaction or reagents, they may also be amplified during the process. This can lead to incorrect results, like false positives. Contamination cannot be removed or reduced once it has occurred, so prevention through proper handling procedures is crucial.

To reduce contamination, PCR experiments are often conducted in controlled environment. This can involve controlled air flow and high-quality filtration mechanisms. PCR is often conducted in a biological safety cabinet but can also be carried out in a laminar flow hood.

Laminar Flow Hoods and PCR

Using a vertical laminar flow hood is a suitable way to prevent contamination. These hoods use HEPA filters to keep the air inside the workspace clean.

While a laminar flow hood is great for working with tissue culture or other sensitive material, it is not safe for handling infectious agents. This hood is designed to keep the samples safe from contamination from the outside using a filtered laminar air flow. Reducing contamination helps to eliminate the risk of false positives or negatives. However, it does not protect the user working with the sample.

In order to conduct PCR in a laminar flow hood, your set-up must include a UV light for sterilization, like the Ossila laminar flow hoods. The built in UV light within our hood enables you to sterilize the workspace before and after each use.

Good Laboratory Practises

Correct use of the laminar flow hood during the PCR process is important. There are several steps you can take to ensure this:

  1. Clean the hood, the equipment inside the hood, and the surrounding area with a disinfectant before and after preparing the samples for PCR.
  2. Turn on the UV light for at least 20 minutes before and after handling a sample, whether it be pre-PCR preparation or post-PCR analysis. This helps to maintain a contamination-free environment.
  3. Wear personal protective gear like a lab coat and gloves. It is also recommended that you use new gloves at each step of the process.
  4. Swap or clean your equipment at each stage. For example, use a different pipette for the aliquoting of PCR reagents, the low copy pre-PCR sample target, and the amplified DNA.

Contributing Authors

Written by

Linda Vidova

Scientific Writer

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