A Cornerstone of Molecular Biology: The PCR Reaction

A Cornerstone of Molecular Biology

Since its invention in the mid-1980s, the polymerase chain reaction (PCR) has been the cornerstone of molecular biology and genetics – almost like a scientist’s bread and butter! PCR acts like a molecular photocopier, producing huge numbers of identical DNA fragments from the original sample. The PCR products, identical to the original target sequence, can then be used to analyse the sequence and identify, for example, mutations in the amplified gene/region.

An Overview of the PCR Reaction

PCR firstly involves making the reaction solution – the PCR cocktail. Once this has been made, it can be put into a PCR machine or thermocycler where the solution is cycled through a number of temperature settings. These different temperatures facilitate each stage of the reaction, and the more cycles you go through, the more product is produced.

Ingredients of the PCR Cocktail

1. Template DNA

A DNA sample with the target sequence in it. This could be from a tumour if you’re looking for cancer-causing mutations, or from any other DNA-containing tissue-type.

2. dNTPs

These are the free base nucletodies that will be used to make our new DNA strands while amplifying the target region.

3. Primers

You need two primers for the reaction – a forward and a reverse primer – that bind each of the two strands of the DNA and act as a starting point for the synthesis of new strands. We’ll explain more about this in a moment.

4. Buffer Solution

This keeps the solution at an optimal pH for the PCR reaction to occur.

5. Polymerase

This is the enzyme that will actually synthesis the new strands of DNA. Polymerases such as Taq are normally used because their activity is not disrupted at high temperatures, meaning that you don’t have to add more polymerase after the high temperature parts of the thermocycle.

6. MgCl2 Solution

This provides the Mg2+ ions needed as a co-factor for the polymerase. Whilst MgCl2 solution is often used, Mg2+ is sometimes included in the buffer solution, so may not need to be added separately. The concentration of magnesium ions also helps determine how fast and how accurately the enzyme will replicate the sequence.

7. Water

To make up the reaction volume.

Primers and Primer Design

Primers are short single strands of DNA the bind at either side of your target sequence to allow amplification of the region they encompass (take a look at the diagram below). They’re usually around 18-30 nucleotides long.

primers2

The primers are designed so that they’re complementary to regions that flank the target gene, so they’ll bind to these sequences (and only these sequences). They’re also designed to incorporate a certain number of C and G nucleotides to alter the temperature at which they’ll attach (anneal) to the DNA.

Thermocycle and Amplification

thermocycle2

Step 1: 90-95oC

This activates the poylerase enzyme.

Step 2: 90-95oC
The denaturation step: the high temperature separates the two strands of the DNA, allowing the other agents in the PCR cocktail to access the genetic information.

Step 3: 55-65oC
The annealing step: at this cooler temperature the primers can bind to the regions next to the target gene that we want to amplify. This binding is crucial, as the primers act as a starting point for the synthesis of new strands that will become the copies of the target region.

Step 4: Around 72oC
The synthesis step: this is the optimum temperature for the enzyme to work, allowing efficient incorporation of the dNTPs by the polymerase to extend the primers into full-length copies of the target region.

Repetition of Steps 2, 3 and 4
The repetition of the separation, primer annealing, and synthesis steps allows more copies to be make. The number of copies of the target region increase exponsentially, as with each cycle the number of total copies is double, as shown in the diagram below.

Step 5: Around 72 oC
This step follow the repetition of steps 2-5 and makes sure that strand synthesis has been completed before the reaction is stopped.

Step 6: 4 oC
This cools the reaction to a temperature at which the reaction stops.

exponential inc2

So there’s a quick guide to the PCR reaction. Remember: all the components of the PCR cocktail are crucial for it to work, primer design is key to specifically amplify the target region, and more cycles means more copies!

References

http://oregonstate.edu/dept/biochem/hhmi/hhmiclasses/bb494/pcrohtwo.html

http://www.seedtechnology.net/docs/PCR%20Tips%20and%20Tricks.pdf

http://www.qiagen.com/knowledge-and-support/spotlight/protocols-and-applications-guide/pcr/

Mullis K, Faloona F, Scharf S, Saiki R, Horn G, & Erlich H (1992). Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. 1986. Biotechnology (Reading, Mass.), 24, 17-27 PMID: 1422010

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