The DNA molecule consists of two intertwined strands which are known as a double helix. It is an extremely complex molecule carrying tens of thousands of genes. Luckily, only relatively small sections of a person’s DNA need to be analysed in order to perform a paternity test. These sections are referred to as microsatellite markers or loci (see The Paternity Testing Process section for more details). The amount of DNA extracted from a sample is small, so before the microsatellite markers can be analysed, they must first be copied (or amplified) millions of times so that there are enough copies present to allow detection. This amplification process is known as a Polymerase Chain Reaction (PCR) and it takes about 4 hours.
But how can the laboratory analyst control which fragments of DNA to amplify? The trick lies in knowing the sequence of the DNA bordering these fragments. Each bordering sequence is unique, meaning that the DNA pattern constituting it is not found anywhere else throughout the entire human DNA. On the other hand, this DNA pattern is identical amongst all humans. Thus, the pair of sequences bordering a microsatellite marker uniquely identifies the start and end of the marker.
For the PCR, specially designed molecules (called primers) are introduced into the purified DNA solution. These primers are single-stranded DNA molecules designed to bind specifically to the unique sequences bordering the microsatellite markers. Single nucleotides, which are the building blocks that constitute DNA, together with an amount of another special molecule, or enzyme, called Taq polymerase, are also added to the mixture.
In the PCR reaction, the double stranded DNA is first denatured by exposing it to a high temperature of around 94 Celsius. At this temperature, the DNA unwinds and the bonds between the two strands of the DNA molecule are broken, leaving two single stranded DNA molecules. The solution is then allowed to cool down, and as this happens, the primers bind to their targets at both ends of the DNA markers. The Taq polymerase recognises the site where the primer ends and starts inserting single nucleotides at this point, building the single strand DNA between the two primers back into double-stranded DNA. This constitutes the end of the first PCR cycle.
Thus, at the end of each PCR cycle the targeted DNA fragments would have doubled in number. For paternity testing the PCR cycle is repeated about 40 times, so that by the end of the PCR reaction there are millions of copies of the required DNA fragments.
DNA Analysis
Once the microsatellite markers have been amplified, they are placed into an instrument called an automatic DNA analyser, which separates the markers by size. The primers used during the PCR are fluorescently labelled. This allows the DNA analyser to detect the DNA markers and measure their size by means of a laser. The size information is fed into a computer, and a special software program analyses the data and assigns a repeat number to each fragment.
The detection and data analysis process takes between 5 and 7 hours and the final result is displayed in graphic form on the computer as show in Example 1 and Example 2. Each microsatellite marker appears as two peaks, one for each complimentary copy. The position of a peak is determined by its size, so if the two genetic complements of a given marker are of equal size they appear superimposed as one peak.
Once this process has been finalized the resulting data is then processed for statistical analysis in order to be able to issue the result of the paternity test.
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