Polymerase chain reaction (PCR),a revolutionary method developed by Kary Mullis in 1983 is now an essentialtechnique used in clinical and research laboratories for a broad variety ofapplications. PCR is an in vitro technique for amplification of a specificsequence of DNA that lies between two regions of known nucleotide sequence(Rastogi, 2003). PCR can detect small amounts of DNA, thus it is possible toanalyse processed and heat-treated food products. PCR is often characterized byhaving quick processing time and low cost (Aida et al.
, 2007). By using DNA polymerase,primers, and nucleotides, PCR reactions will amplify the target nucleic acidsequences. The template for a PCR reaction may be any nucleic acid sequence ofinterest, and the nucleic acid source may be DNA, RNA, or cDNA. Primers areshort sequences of nucleotides that are synthesized in vitro designed to annealto opposite strands of a specific nucleic acid template target. The polymeraseenzyme will add the deoxyribonucleoside triphosphates (dNTPs or nucleotides)onto the ends of the primers to extend the nucleic acid chain based on thetemplate sequence, effectively doubling the amount of that DNA segment (Walker-Daniels, J., 2015).
As each consecutive cycle doublesthe amount of DNA synthesized in the previous cycle, this will result in theexponential accumulation of the specific target fragment (Saiki et al., 2000). An effective detection of a species by PCR isdependent on the selection of the specific DNA target template. MitochondrialDNA (mtDNA) sequences were commonly used as it offers a series of advantagesover other genetic markers like cell nucleus DNA. The advantages ofmtDNA in lies in its high number of copies per cell and its genes evolve muchfaster than nuclear ones.
Therefore it contains more sequence variation which easesthe identi?cation of phylogenetically related species, for instance, betweencow and goat or differentiating between the different species of deer (Fajardo et al., 2007; Pereira, Carneiro, & Asch,2010). 184.108.40.206 Inhibition of Polymerase Chain Reaction The amplification of DNAextracted from samples by the PCR can be limited by the presence of inhibitorycompound (de Boer et al., 1995).
The inhibitory compound maybe co-extracted from the sample or introduced during sample processing ornucleic acid extraction, leading to disruption of the PCR amplification process(Opel, Chung, & Mccord, 2009; Syarifah NurSyakira, 2016).The major repercussion of inhibition of the PCR is a decreased sensitivity orfalse-negative result (Schrader et al., 2012). Pharmaceutical excipientscan also be an inhibitor as some might have adsorbent properties that can trapthe DNA compromising the subsequent amplification process (Costa et al., 2015).
The potential mechanisms of PCRinhibition could be binding of the inhibitor to the polymerase or interactionof the inhibitor with the DNA. Another potential mechanism could also beinteraction with the polymerase during primer extension (Opel et al., 2009). Organic and inorganic substances,which may be dissolved or solid, can act as PCR inhibitors.
PCR inhibitors canbe found in a variety of biological materials (e.g. blood, tissue, body fluid),fabric, soil, and food (e.g. meat, milk, seafood). A list of common inhibitorysubstance is listed in Table 1.
3. Other than that, inhibitory substances mayalso be accidentally added during transport, sample processing or nucleic acidextraction.