Question

Explain the technology of polymerase chain reaction (PCR) and how it applies to forensic
DNA typing

Answer 1

PCR is a technique used in the lab to make copies or amplify a particular section of DNA. It was first developed in the 1980s.

Five core requirements are needed to set up a PCR.

1. The DNA template to be copied
2. Primers, short stretches of DNA that initiate the PCR reaction, designed to bind to either side of the section of DNA
3. DNA nucleotide bases. DNA bases (A, C, G and T) are the building blocks of DNA and are needed to construct the new strand of DNA
4. Taq polymerase enzyme to add in the new DNA bases
5. Buffer to ensure the right conditions for the reaction.

PCR involves a process of heating and cooling called thermal cycling which is carried out by machine.

There are three main stages:

1. Denaturing – when the double-stranded template DNA is heated to separate it into two single strands. During this stage the cocktail containing the template DNA and all the other core ingredients are heated to 94-95⁰C. The high temperature causes the hydrogen bonds between the bases in two strands of template DNA to break and the two strands to separate. This results in two single strands of DNA, which will act as templates for the production of the new strands of DNA. It is important that the temperature is maintained at this stage for long enough to ensure that the DNA strands have separated completely. This usually takes between 15-30 seconds.
2. Annealing – when the temperature is lowered to enable the DNA primers to attach to the template DNA. During this stage the reaction is cooled to 50-65⁰C. This enables the primers to attach to a specific location on the single-stranded template DNA by way of hydrogen bonding. Primers are single strands of DNA or RNA sequence that are around 20 to 30 bases in length. The primers are designed to be complementary in sequence to short sections of DNA on each end of the sequence to be copied. Primers serve as the starting point for DNA synthesis. The polymerase enzyme can only add DNA bases to a double strand of DNA. Only once the primer has bound can the polymerase enzyme attach and start making the new complementary strand of DNA from the loose DNA bases. The two separated strands of DNA are complementary and run in opposite directions (from one end - the 5’ end – to the other - the 3’ end); as a result, there are two primers – a forward primer and a reverse primer. This step usually takes about 10-30 seconds.
3. Extending – when the temperature is raised and the new strand of DNA is made by the Taq polymerase enzyme. During this final step, the heat is increased to 72⁰C to enable the new DNA to be made by a special Taq DNA polymerase enzyme which adds DNA bases. Taq DNA polymerase is an enzyme taken from the heat-loving bacteria Thermus aquaticus. This bacteria normally lives in hot springs so can tolerate temperatures above 80⁰C. The bacteria's DNA polymerase is very stable at high temperatures, which means it can withstand the temperatures needed to break the strands of DNA apart in the denaturing stage of PCR. DNA polymerase from most other organisms would not be able to withstand these high temperatures, for example, human polymerase works ideally at 37˚C (body temperature). 72⁰C is the optimum temperature for the Taq polymerase to build the complementary strand. It attaches to the primer and then adds DNA bases to the single strand one-by-one in the 5’ to 3’ direction. The result is a brand new strand of DNA and a double-stranded molecule of DNA. The duration of this step depends on the length of DNA sequence being amplified but usually takes around one minute to copy 1,000 DNA bases (1Kb).

These three processes of thermal cycling are repeated 20-40 times to produce lots of copies of the DNA sequence of interest. A complete PCR reaction can be performed in a few hours, or even less than an hour with certain high-speed machines. After PCR has been completed, a method called electrophoresis can be used to check the quantity and size of the DNA fragments produced.

DNA profiling (DNA typing, genetic fingerprinting, DNA testing) is a technique used by forensic scientists to identify someone based on their DNA profile. PCR can be used as a tool in genetic fingerprinting. This technology can identify any one person from millions of others. For example, tiny samples of DNA isolated from a crime scene can be compared with DNA from suspects, or compared with a DNA database. Such procedures can identify or rule out suspects during a police investigation. PCR-based DNA fingerprinting can also be used in parental testing in which an individual is compared with their close relatives and the actual biological father of a child can be confirmed or ruled out.