GENETICS

Genetics

Genetics

The polymerase chain reaction is utilised by a broad spectrum of researchers in an ever-increasing variety of technical disciplines. In microbiology as well as molecular biological research, for demonstration, PCR is utilised in study laboratories in DNA cloning methods, Southern blotting, DNA sequencing, recombinant DNA expertise, to title but a few. PCR is very useful in clinical microbiology laboratories for the diagnosis of microbial diseases and epidemiological studies. PCR is furthermore utilised in forensics laboratories and is particularly helpful for the reason that only a minute allowance of initial DNA is needed, for demonstration, adequate DNA can be got from a droplet of body-fluid or a lone hair.

The polymerase chain reaction (PCR) has become the method of choice for most DNA testing applications, including disease diagnosis, species identification or genetically modified (GM) food analysis. This technique is based on the logarithmic amplification of a specific target DNA sequence to produce detectable quantities of DNA. With the introduction of real-time PCR platforms, analyses have quickly changed from qualitative (yes/no) to quantitative tests. Real-time PCR monitors the increase in target DNA throughout the reaction using fluorescence chemistries: the higher the fluorescent signal, the more PCR product has been synthesised. A CT value is assigned for each amplified target. This is the PCR cycle at which the fluorescent signal passes a defined threshold. This CT value allows the analyst to calculate how much of the target sequence was present in the starting material with relative ease. However, the CT value is a factor of both starting copy number and amplification efficiency, and the effects of amplification efficiency are often overlooked.

Maintaining Pcr Efficiency

PCRs rarely amplify a target sequence at 100% efficiency and a decrease in efficiency will lead to an under-estimation of the target copy number. PCR efficiency is not only affected by poor primer design or poor reaction optimisation, but also by inhibitors co- extracted with the DNA. These inhibitors can disrupt the PCR in a number of ways, including binding to the DNA or polymerase enzyme, mopping up the free magnesium ions from the buffer or changing the pH or viscosity of the reaction. A different suite of inhibitors can be present, depending on the matrix the DNA was extracted from, as well as the choice of extraction technique. When too much inhibitor is present in the extracted DNA the PCR will fail completely, potentially producing a false negative result. The efficiency of the PCR amplification can be calculated using a dilution series of the extracted DNA. A one-in four dilution series should produce results with each CT value separated by two points. This approach is suitable for method validation but not for routine analysis.

Testing laboratories generally prepare in-house DNA controls to use with routine assays if these controls are not commercially available or included in a test kit. Traditionally, DNA controls are prepared by measuring the concentration of a DNA solution using absorbance at 260nm and then diluting the solution to obtain the desired concentration or even target copy ...