The polymerase chain reaction is a technique which has revolutionized molecular biology since its development in the early 1980s. It allows researchers to amplify small amounts of DNA to quantities which can be used for analysis. Some of the uses to which PCR has been applied include :
- Disease diagnosis, where the causative agent of a disease is identified by its DNA. This is particularly useful when disease agents are difficult to grow in culture or are present in low numbers in a sample
- Forensic investigations, where trace amounts of DNA found at crime scenes (eg. in hair, tissue or body fluids) may be amplified up to a level which allows them to be analysed using methods like DNA profiling. PCR may also be used in other areas where the amount of DNA recovered is vanishingly small or damaged (eg. archaeology)
- Genetic engineering, where genes are introduced into new species – to do this, the genetic material to be introduced must be of a sufficient quantity to ensure efficient transformation of the host cell
PCR relies on a number of characteristics of the DNA molecule:
- The structure of DNA consists of two strands of chains of molecules called nucleotides. Each of these nucleotides consists of a phosphate group, a sugar (deoxyribose) and one of four nitrogen containing bases (adenine, thymine, guanine or cytosine). Each strand runs in the opposite direction to the other (ie. they are anti-parallel), with only certain bases lying opposite each other (an adenine is always opposite a thymine and a guanine is always opposite a cytosine). This means that the two strands are complementary to each other in the order of their bases.
- The two strands of DNA are held together by hydrogen bonding between nucleotide base pairs. Hydrogen bonds are much weaker than the covalent bonds which link individual nucleotides within a strand and may be disrupted by heating the DNA. Therefore, we can separate the two strands of DNA without breaking the DNA strands down by heating to around 95°C – this process is called denaturation.
- Primers are short sequences of complementary DNA which bind to certain nucleotide sequences along the DNA strand. They tend to bind onto the single DNA strands at higher temperatures than the entire complementary strand. This means that if the temperature is cooled from 95°C to around 50-60°C, the primers will bind to the single strands before the complementary whole strands do. This process is called annealing.
- The production of a new complementary strand of DNA using a single strand is performed by a class of enzymes called polymerases. These enzymes start off by binding to the primers and then extend the primers by adding new nucleotides to the 3’ end, using the single stranded DNA as a template.
- Most polymerases function best at the temperatures that their cells operate at (eg. 37°C for human cells). However at this temperature, most of the entire complementary strands will also reattach and interfere with the function of the polymerase (in cells, the DNA strand is kept unwound during replication by enzymes such as helicase). There are some organisms which operate at much higher temperatures – Thermus aquaticus is a bacterium which lives in boiling hot springs. The polymerases which it uses operate best at around 72°C. Therefore these enzymes may be used to ensure that the strands are kept separate during the extension process.
A PCR technique needs the following reagents :
- A DNA sample which acts as the template on which the new DNA will be built
- 4 deoxyribonucleoside triphosphates (adenosine triphosphate, guanosine triphosphate, thymidine triphosphate and cytidine triphosphate) – these are the “building blocks” from which the new DNA molecules will be made
- Taq Polymerase, or similar polymerase enzyme, which operates best at high temperatures
- 2 Primers (forward and reverse) to start the process of replication. These primers are designed to be complementary to the nucleotide sequences at the beginning and the end of the section of DNA we want to amplify
- Buffers and salts to create the correct conditions for the enzyme to function
A lot of work has to go into designing the primers. Firstly, we need to know the sequence of the section of DNA we are wanting to amplify, particularly the “beginning” (5’) and “ending” (3’) of the sequence. The primers need to be designed so that they are complementary to a unique sequence of nucleotides “upstream” and “downstream of the sequence of interest. They cannot match a sequence within the area of interest (or the PCR will start off too late and miss a portion of the area we want to amplify), and they should also not have complementary regions within themselves (or they will fold over and bind to themselves, forming a “hairpin”. Lastly, the forward and reverse primers should not be complementary, or they will anneal to each other and form a “primer dimer”. We can avoid most of these problems using primers of 15-20 nucleotides in length (note that the examples in the diagrams below use 5 nucleotide primers for simplicity – we would not use these in a real PCR reaction.
Different protocols need to be developed for each PCR procedure, depending on the primers used, the length of template DNA or the type of polymerase involved. However each protocol has the following basic steps :
These steps are repeated between 25 and 35 times, with the amount of DNA roughly doubling each time. This might not seem like much, but after 35 cycles, one DNA molecule could theoretically yield in excess of 34,359,738,370 molecules (235).
When PCR was first developed, scientists had to change the temperature manually, swapping the samples between waterbaths kept at just the right temperature. However, now they use PCR cycler machines which heat and cool samples precisely and automatically.
You might also want to listen to Biorad’s “PCR song” (note : this opens to You Tube – the site may not be available to some users).