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The polymerase chain reaction (PCR) has become a vital tool within biotechnology, genetic research forensic science and medicine since it was first invented by Kary Mullins in 1983 (Hongbao, 2005). It has revolutionised all aspects of science as most DNA profiling methods use the polymerase chain reaction (Gunn, 2006). PCR is an extremely fast and efficient process taking only a few hours in contrast with other cloning techniques that can take days (Klug et al, 2009). PCR is the process of amplifying a specific region of DNA. Once DNA has been extracted from the evidential material, this may be biological samples; it is put through a series of temperature controlled conditions. The process consists of three basic steps, denaturation, annealing and extension (Goodwin et al, 2007).
The polymerase chain reaction.
The first stage of the process is to separate the double stranded DNA template (see fig 1, page 3, denaturing process). This is done by incubating the sample at 94°C - 95°C. At this temperature the hydrogen bonds between the nitrogenous bases are broken down causing the strands of DNA to separate into single strands (Russell, 2006). The temperature is next reduced to 50°C - 60°C allowing the primers to 'bind' (fig 1, annealing process) to their complementary sequences of the DNA template (Gunn 2006). The temperature is raised to 70°C - 75°C in the third stage (fig 1. Extension process). Making a double strand of the target DNA is achieved by the taq polymerase extending the primers and adding nucleotides to the complementary sequences in the 5' to 3' direction (Klug et al, 2009). Taq polymerase is a thermal stable polymerase that is derived from the bacterium Thermus aquaticus, and has an optimum temperature of 80°C (Chien, 1976). The procedure is repeated from 28 to 32 cycles (Goodwin et al. 2007). It is not until cycle 3 that the process produces unit length double stranded DNA, until this point only the product between the primers will increase exponentially. In order for the polymerase chain reaction to work there are several essential components needed (see table 1 below).
Table 1. The essential components of PCR. Table information was taken from Butler, forensic DNA typing and edited (see Reference page 6).
Very specific DNA sequence to be copied (amplified)
Define the region to be analysed
Thermostable DNA polymerase
Taq DNA polymerase
Stabilises the interactions and is needed for the taq polymerase to function
Incorporated into the template DNA strand during replication
Maintains ph. and salt conditions during the reaction
Figure 1. Shows the three basic steps of polymerase chain reaction. (Image taken from virtual medical centre website).
PCR Inhibition and Limitations.
There are certain chemicals that will affect or inhibit the Taq polymerase such as blood, soil samples, tissues and fabrics (Bessetti, 2007). The blue dye from denim is a known inhibitor, and as denim is a very popular material scientists have had to accommodate this problem by adapting extraction processes. Methods have been developed, such as silica binding, during the extraction process to remove common inhibitors (Goodwin et al. 2007).
Although the benefits of PCR are extremely useful to the science community there are some limitations. Certain sequences of the target DNA must be known and there are contamination issues (Klug et al, 2009). Due to such a small amount of DNA being able to be used in PCR the contamination risk are significantly higher.
What is so special about PCR?
The technology has allowed us to analyse evidential material that previously could not be analysed. It has allowed us to replicate fragments of DNA sequences that can be further analysed through DNA profiling. In any situation where DNA may be used, a DNA profile is created. DNA profiling is one of the most reliable and conclusive methods of personal identification available today (Butler, 2005). The structure of DNA is unique to an individual just as a fingerprint is.
The sensitivity of the PCR reaction has made it possible to generate a DNA profile from only a few cells. Degraded samples can be analysed as such a small amount of cellular material is needed for the reaction (Goodman et al, 2007). Cases left unsolved in the past are now being reopened and re-examined using this technology.
Databases have been compiled with DNA profiles. In the United Kingdom the National DNA Database (NDNAD) is used, in the United States Combined DNA Index System (CODIS). The DNA sequences are compared against known sequences (Goodman et al, 2007). The most commonly used are short tandem repeats (STRs).
Figure 2. Shows the CODIS core short tandem repeats (STR) markers.
Other markers used include single nucleotide polymorphisms (SNPs) and variable number tandem repeats (VNTRs).
Evidence and Court.
Another important use of DNA profiling is in the court system. A DNA profile can be used to establish paternity in custody and child support legal issues (Goodwin et al, 2007). This technique can also be applied to sexual assault cases, where the DNA evidence found can be compared and connected back to the suspect (White, 2005).
The First DNA Case.
In 1988 Colin Pitchfork was sentenced to life imprisonment for the rape and murder of two young girls (Gunn, 2006). This was the first case that used DNA profiling in a forensic investigation. The semen sample taken from the girls at the crime scene was later found to be a match from the suspects (Gunn, 2006). This case is famous for not only proving that Colin Pitchfork was guilty, it also proved that a previous suspect who had come forward and confessed to the crime, was not, in fact, telling the truth. DNA evidence cannot lie.
DNA profiling is used to analyse and diagnose inherited disorders in humans, old, young, in pre-natal and new-born babies (Russell, 2006). This has had a profound effect on medical research.
Distinguishing between species.
PCR has been used with identifying different species which include plants and animals. Narayanan et al, (2006) used PCR to find inter simple sequence repeat (ISSR) markers in Tectona grandis (teak). They used four different extraction methods to evaluate genomic DNA. This type of information can be used at crime scenes to prove that bodies have been moved after the initial crime (White, 2005). Plants that grow is specific areas have become most useful in forensic botany cases.
There is no doubt that the polymerase chain reaction is has revolutionised the way science has used and analysed DNA evidence. The power to discriminate between samples and the sensitivity it possess are essential. The medical research done and currently being studied with genetic disorders will help us to understand why we inherit certain diseases and hopefully one day will cure them.