The Molecular biologist’s tool belt: PCR

The polymerase chain reaction (PCR) and the subsequent development of the thermocycler – the machine that carries it out – has revolutionised the use of genetics in the lab. Simply put, PCR takes a small amount of template DNA, and repeatedly copies a small section to create a much larger amount of this small section. Almost every protocol in molecular biology at some point will require some amount of PCR, so knowing the basics can be invaluable. On top of the near ubiquitous nature of the technique, there are also several more advanced versions that can be used analytically, greatly increasing the scope of the technique.

So whilst you might be a PCR newbie or a master of reverse-transcriptase-real-time-quantitative-multiplex-PCR, this article should be of some use to you. In the first half we’ll take a look at the fundamentals, before moving on to some of the interesting applications in microbiology and beyond.

The Fundamentals

The PCR reaction has 3 main steps. First, DNA is melted – the two strands separated at a high temperature to create single stranded DNA. This is followed by a lowering of temperature called the annealing step, which allows DNA primers to anneal to the template strand. Following this the temperature is raised again to the extension stage, where the polymerase actually copies the DNA. Some of these terms might be unfamiliar, but just hold on tight, all will be explained.

Polymerase_chain_reaction.svg

This diagram outlines the reaction – steps 1-3 will be repeated many times to achieve exponential amplification

So to get started with PCR you need several components:

  • Template DNA
  • Primers
  • DNA Polymerase
  • Nucleotides
  • Reaction Buffer

Let’s look in more detail at each of these.

Template DNA

This can be DNA in any form – genomic DNA extracted directly from an organism, a gene in a plasmid, even another PCR product. As long as it contains the gene you’re trying to clone, the template can be any length. Some PCR protocols use RNA rather than DNA, but more on this later.

Primers

I mentioned primers earlier, but didn’t explain what they were. Primers are short lengths of DNA (usually around 20 bases) that act as the initial templates for the polymerase to go off of. They also denote where replication should start. The primers are complimentary to the template – and you need one in the 5’ and 3’ direction to make sure you copy both strands.

There are various tools online to help design primers, and I could write pages on the top tips, but in my personal experience, it didn’t matter how well I read up on the best primer design or how many notes I took in classes in my undergraduate and masters, the best resource for designing a primer was always the post-doc in my lab. Primer design can be very nuanced and so an experienced eye goes a long way – maybe someday you’ll be that experienced eye too.

The really great thing about primers is that they don’t have to be complimentary for their whole length – meaning you can add portions of DNA to help in cloning, in particular restriction endonuclease recognition sites and antibody tags. Since the polymerase reaction repeats many times, the extra DNA on the primers becomes copied in and amplified to the final PCR product.

DNA Polymerase

The key to this polymerase is that it is thermophilic, enabling the reaction to be carried out at high temperatures. The standard polymerase is called Taq – as it was first isolated from Thermophilus aquaticus – but there are many different polymerases available for different needs. Some are high fidelity, enabling more accurate PCR amplifications, others are error prone to be used for mutagenesis, some are able to stay on the DNA longer for longer amplifications, and so on.

Nucleotides

This one is fairly self-explanatory: to replicate DNA, you need DNA bases. Using nucleotide triphosphates provides the energy for DNA synthesis (just like in the cell) so using these is key.

Reaction Buffer

Most polymerases come with a reaction buffer that’s optimised for the polymerase, but the key components of the buffer are salts and magnesium – the polymerase can’t operate without them. Some polymerases will come supplied with two buffers, one for standard amplifications and one for GC rich samples. GC rich samples can pose a problem to polymerases, so if your DNA sequence is particularly rich in GC (often the case when working with a thermophilic organism), these buffers can be a lifesaver.

thermocycler

The thermocycler has made PCR routine – no more running between water baths at different temperatures for hours!

So now you have everything you need to get started, the only thing left is the protocol. Of course the standard practise varies from lab to lab, and you have to design your protocol to your template and primers, so this is another one of those topics that could fill a textbook. In short the melting temperature is usually set to around 95°C, the annealing temperature is governed by your primers, and your extension temperature by your polymerase. Similarly, your extension time will be detailed by your polymerase and is to do with the length of the fragment you’re amplifying, and your annealing and melting times will alter slightly with the primers and template you’re using.

The final thing you need to decide is the number of cycles. The PCR reaction repeats many times to achieve exponential amplification, and typically at least 30 cycles are used. It really depends on how much DNA you want at the end of it.

 

Some More Applications

If you’ve already got a solid understanding of PCR, then the fundamentals above will be quite familiar to you. But even to someone who has spent years doing PCR, there are some applications that will be new to you. There are many different types of PCR, and I touch on just a small cross-section of them here, and is by no means exhaustive, but this should be a varied enough collection to show the versatility of a technique that on the face of it just amplifies DNA.

Touch-down PCR

Whilst this isn’t much of an advanced technique, it is a useful one when your reaction just isn’t working. PCR always requires some trial and error, but sometimes this can be fruitless, and playing around with the temperature just won’t work. Touch-down PCR is a great technique that circumvents this, and works by starting a few degrees above the predicted annealing temperature of the primers and slowly stepping down the temperature with each cycle until it is a few degrees below the predicted annealing temperature. This technique is also great if you are getting a lot of background noise from non-specific binding in your sample, as the higher start temperature reduces non-specific interactions.

Reverse Transcriptase PCR

I mentioned earlier that you may wish to use RNA as the template for PCR to create cDNA. In this, reverse transcriptase is used to convert the RNA into cDNA, and a polymerase then continues with the usual PCR protocol on this cDNA. This might be done as separate steps, but can also be done all at once by including both enzymes and all primers in the same reaction tube.

Inverse PCR

Inverse PCR is a technique not used to amplify DNA, but to find out more information about its location in a longer sequence. By placing primers that run in the opposite direction to usual, the flanking DNA sequence on the target DNA is amplified. By then ligating then analysing these products, details on the region surrounding the DNA of interest are found. Therefore, it is particularly useful at looking at where transposable sequence elements or viruses have inserted DNA, by using primers complimentary to the transposon or viral sequence to gain information about the insertion site.

Real Time (Quantitative) PCR

This technique uses fluorescence to measure the amount of DNA produced in real time. The fluorescence markers might be non-specific and detect double stranded DNA (i.e. they only bind in the annealing and extension phases) or be short probes with fluorescent tags which fluoresce only when bound to its complementary sequence (only binding in the annealing phase as they are removed by the polymerase during extension). The use of fluorophores allows a direct quantification of the sample DNA and the PCR products, making this technique very useful in diagnostic settings.

Multiplex PCR

Multiplex PCR uses many different primers to amplify different regions of the target sequence at once. This is useful for high throughput analysis and in diagnostic samples, as it vastly increases the amount of information from one run of PCR. Various kits are available to help with this, including fluorescent markers that help differentiate the products.

Digital PCR

Whilst this might sound like an in silico technique, this is actually an analytical technique used to measure the number of occurrences of a target DNA sequence within a DNA sample. This is done by partitioning the template DNA into many smaller sections, and then running PCR on each of these. Using some statistical methods, the number of reactions that did not amplify compared to the total number of reactions can be used to estimate the number of times a short sequence occurs in a larger sample. This can be very useful for estimating copy number variants or point mutations. The ‘digital’ name is because rather than measuring the amount of PCR product to estimate the occurrence of a sequence (a less accurate method), this technique simply measure the presence or absence of a reaction, creating a binary scale.

As you can see, from fundamentals to advanced applications, PCR is one of the cornerstones of molecular biology and the biological sciences. A firm rooting in this technique will get you a long way, so brush up where you can, as you never know when the next gene of interest is going to need amplifying.

 

Robert G Millar (University of Warwick)

Society for Applied Microbiology



Categories: Feature Articles

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