The Polymerase Chain Reaction (PCR) was invented in 1983 by the biochemist Kary Mullis and has since become a fundamental part of many procedures used in genetic testing.
PCR is an amplification technique, the use of which enables millions (or even billions) of copies of a specific DNA sample to be created. These copies may be of the entire DNA sample, or a specific sequence of interest. Amplification is important as it allows scientists to take a small DNA sample and amplify it to a large enough amount to study in detail. Without amplification DNA testing on many samples would not be possible.
PCR methods rely on thermal cycling, which is the exposure of samples to repeated cycles of heating and cooling to permit temperature dependent, enzyme driven reactions. For each of these “temperature cycles”, the double stranded DNA is opened up creating 2 single stranded DNA sequences in a part of the process known as “denaturation”. These single stranded DNA sequences are then both converted to double stranded DNA sequences in a part of the process known as “elongation”. The end result is that the total number of DNA strands is doubled with each cycle. As PCR comprises of multiple cycles, this technique allows for a huge amplification of the amount of DNA available to be tested.
Which DNA sequences are amplified depends on the primers used during PCR. Primers are complimentary short sequences that bind to the single stranded DNA sequences and start the process of elongation. These can either be universal, meaning that PCR amplifies an entire DNA sample, or targeted, meaning that they amplify a specific sequence of interest.
Yourgene Health PCR based kits fall into 2 different categories dependent on which method of PCR they use. Both methods rely on the detection or lack of detection of a sequence of interest after amplification to show the presence or absence of said sequence.
Amplification Refractory Mutation System (ARMS)
Amplification Refractory Mutation System (ARMS) is a PCR based technique that is used to detect small mutations in DNA sequences of interest. It uses sequence specific primers that allow for the amplification of a DNA sample only when a target sequence is present. A target sequence could be anything but is most commonly a known disease-causing mutation.
This means that amplification is an indication of the presence of a target sequence (potentially a disease-causing mutation). Likewise, a lack of amplification is an indication of the lack of a target sequence, telling us that said sequence (potentially a disease-causing mutation) is not present within a sample.
Specialist capillary electrophoresis equipment is used to determine whether amplification has occurred after PCR. This is possible as the primers used in ARMS are fluorescent, meaning that amplified DNA sequences are also fluorescent. The specialist equipment applies light to a sample following PCR and measures the fluorescence, with high fluorescence being an indication that amplification was successful and that the target sequence (potentially a disease-causing mutation) is present in the sample.
Yourgenes Cystic Fibrosis, DPYD and the Thrombosis Risk Panel kits, amongst others use ARMS technology.
Quantitative Fluorescent PCR (QF-PCR)
Quantitative Fluorescent PCR (QF-PCR) is another PCR based technique used to detect chromosomal aneuploidy. Chromosomal aneuploidy being when the total number of a specific chromosome is not 2 as expected. In QF-PCR, specific markers present on chromosomes of interest are amplified by PCR. The chromosomal copy number is determined by specialist capillary electrophoresis equipment.
QF-PCR also relies on fluorescent primers and measures fluorescence as a measure of amplification. From this, copy number is determined by assessing the fluorescence values of each marker generated by the specialist capillary electrophoresis equipment, and using the fluorescence values of each marker to determine how many copies of the chromosome containing the marker are present in a sample.
In a nutshell, if the fluorescence value of the chromosome 21 marker is around 150% of the fluorescence value of the other chromosomes, then there are probably 3 copies of chromosome 21 (Down’s syndrome) in the sample.
The Yourgene Health QST*R range, QST*R Pregnancy Loss and Male Factor Infertility kits uses QF-PCR technology.