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PCR and other methods

How do they do that? A quick overview of the principle.
Tools needed.
Making Choices : which primers, which temperature?
Let's do the maths!
Applications and disasters.

How do they do that? A quick overview of the principle.

Let's start at the beginning. We have a solution of DNAask Dr Chromo!. If we could see the molecules, we would see the famous double helixask Dr Chromo!. Here it is shown pulled out flat like a ladder.

All the fragments are double stranded, and they vary a lot in size and sequenceask Dr Chromo!.

Let's concentrate on only one of these fragments.

By submitting DNA to a high temperature, it is denatured. i.e. separated into single strands.
If we mix this solution of DNA with some short sequences (16-26 nucleotides) of single-stranded DNA (called primersask Dr Chromo!), and cool down the solution slowly, the primers hybridise (stick) to the fragment of DNA in the solution that they are complementary to. They are are shown in green on the diagram.

What we have now is single stranded DNA with primers bound to it.

If we incubate the DNA and primers with a DNA polymerase (Taq polymerase) and the necessary nucleotidesask Dr Chromo!, the polymerase will start polymerising DNA from the template in one direction only (the polymerisation of DNA is allways from 5' to 3') starting at the primers (the polymerase will not start if there are no primers).

The polymerase will start adding nucleotides to the primer (shown in green) and go up to a distance of 7 kbask Dr Chromo! depending on the conditions of the reaction.

After this first cycle, most of the DNA is still single-stranded, but on each strand, after the primer, there is a certain length of double-stranded DNA. This length depends on the conditions of the reaction. Taq extends beyond the second primer site

When the cycle repeats, there are twice as many places where the primer can stick assuming the template was copied from each primer binding site to the second primer site.

Among the four single stranded DNAs that we now have, two are the original ones: they are very long (in black on the diagram) and two are the copies. They are not as long as the template DNA because the Taq polymerase started from the primers, and copied only in one direction, on a distance depending on the length of time allowed for the reaction (these copies are in blue in the diagram).

The primers will hybridise to their target sequence and the Taq will polymerise DNA starting from the primer until the end of the template they have stuck onto, or if the template is the original fragment to a distance depending on the lenght of the elongation time. If a primer has stuck on one of the copies from the first cycle, then the new fragments created will have a length equal to the distance between the two primers.

If the two primers are not too far apart, then the fragment of DNA between the two primers will be copied a lot of times. As the length between the primers is constant, all the fragments will have the same size. There will be enough DNA to see as a band on an agaroseask Dr Chromo! gel.

All we need to do to amplify the DNA is submit a solution of DNA, primers and polymerase to cycles of

Animation



Tools needed

A few years ago, it was necessary to manually change the temperature of a dry block, (or use several water baths / blocks) and to add new enzyme after each denaturation because the enzyme was inactivated by the high temperature.

Technology soon solved one of the problems with an apparatus called a thermal cycler, that would change the temperature very quickly. This left the problem of the enzymes. Most enzymes used in molecular biology are extracted from micro-organisms that live at normal temperatures (26 - 37 °C) so their enzymes tend to be optimised to work at these temperatures. Enzymes are proteins, and therefore are destroyed by excessive heat. PCR could not go far without a drastic advance. Fortunately some bacteria live in hot springs. The temperatures there are close to 100°C. If micro-organisms can live there, then their metabolism must be adapted to those conditions. It also means their enzymes are, somehow, protected from heat denaturation. The conditions of life are very difficult in the hot springs in Yellowstone Park, in the USA, but the bacterium Thermophilus aquaticus (Taq), manages to live there.

The discovery of the Taq polymerase (DNA polymeraseask Dr Chromo! extracted from Thermophilus aquaticus), revolutionised PCR. It is now a method, widely used in many labs around the world.



Making Choices : which primers, which temperature? Which methods?

Although PCR seems a very simple method, it requires fine tuning. A lot can go wrong!

The amount of template DNA

In order to obtain clear bands on the final gel, one has to use small amount of DNA, because non-specific hybridisation could render the result ambiguous. One needs to use enough DNA, especially when it is extracted from a small sample. The right amount is found by trial and error.

Primersask Dr Chromo!

Primers are synthesised artificially. They are between 16 and 26 nucleotides long in most cases and are copies of known sequences flanking the area we want to amplify. The amount of GC, and AT in the sequence will determine the annealing temperature of the primer (Why? Think of hydrogen bonds!), and the temperature at which the primer will be denatured. Depending on the kind of PCR we want to perform, we can choose the length and sequence of the primers. There is some software available to help scientists design their primers depending on their type of research.

Cycle and temperature

A normal PCR cycle includes

One can improve the PCR result by adjusting these three temperatures and the length of time at each temperature.

The number of cycles used is usually between 20 and 35, and should not be more than that. After 35 cycles, the Taq polymerase will be damaged. As Taq commits errors when copying, increasing the number of cycles after 35 will only lead to more errors and their amplification.



Let's do the maths

Let's start from one single template and work out how many fragments will be obtained after 30 cycles.

After each cycle, each strand has given rise to a double strand: the number of copies has doubled! After 30 cycles the theoretical number of copies will be 2 to the power 30, which is 1,073,741,824 copies.



Applications and disasters

The PCR method soon found a lot of applications. Nowadays most molecular biology papers quote its use and numerous similar methods have been designed.

Finding a needle in the haystack!

If a researcher works on a gene in an organism that has been sequenced (at least partially) in another organism, they can use some of that known sequence as primers to amplify the gene in their organism.

PCR has medical applications too. Some research is being undertaken to use it as a test to detect HIV DNA in blood (to find out if somebody has contracted HIV, before symptoms appear).

Sequencing small amounts of DNA

There are some kits that enable one to sequence a DNA fragment and which are based on a PCR reaction. The fragment to be sequenced is amplified and sequenced in few steps. Because it involves a PCR step, there is no need for large amount of DNA. This is described in more detail in the chapter on sequencing.

Looking for mutations.

Genetic diseases are caused by mutations of one or more genes. PCR provides a quick way to identify the individuals in the population who have a particular mutation. One particular method is to use three primers. Two of them will amplify a large fragment in both the normal and the mutant individual. The third primer will hybridise in the middle of that fragment, and is characterised by the fact that it's 3' end corresponds to the mutation affecting the mutant individual. Only the mutant will amplify a fragment starting from that primer, and therefore will show two bands on the gel. These methods enable one to screen whole populations for a mutation in one reaction. Pretty powerful.

Disasters

Well, PCR is a very powerful technique! But it can go wrong. For example, if the samples are contaminated with the target sequence, you can amplify the contamination and obtain a false positive, a positive result where you would expect a negative result. A sample of water is always run simultaneously to test that there is no contamination (this is called a control). Extreme caution must always be taken throughout an experiment to prevent unwanted DNA (from the skin of our fingers, for example) from getting into the sample. Internal controls are also perform to diagnose false negatives: abscence of bands due to a failed PCR where a band should exist.

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