Polymerase nucleic acid source may be DNA, RNA, or

Polymerase chain reaction (PCR),
a revolutionary method developed by Kary Mullis in 1983 is now an essential
technique used in clinical and research laboratories for a broad variety of
applications. PCR is an in vitro technique for amplification of a specific
sequence of DNA that lies between two regions of known nucleotide sequence
(Rastogi, 2003). PCR can detect small amounts of DNA, thus it is possible to
analyse processed and heat-treated food products. PCR is often characterized by
having quick processing time and low cost (Aida et al., 2007).

 

By using DNA polymerase,
primers, and nucleotides, PCR reactions will amplify the target nucleic acid
sequences. The template for a PCR reaction may be any nucleic acid sequence of
interest, and the nucleic acid source may be DNA, RNA, or cDNA. Primers are
short sequences of nucleotides that are synthesized in vitro designed to anneal
to opposite strands of a specific nucleic acid template target. The polymerase
enzyme will add the deoxyribonucleoside triphosphates (dNTPs or nucleotides)
onto the ends of the primers to extend the nucleic acid chain based on the
template sequence, effectively doubling the amount of that DNA segment (Walker-Daniels, J., 2015). As each consecutive cycle doubles
the amount of DNA synthesized in the previous cycle, this will result in the
exponential accumulation of the specific target fragment (Saiki et al., 2000).

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An effective detection of a species by PCR is
dependent on the selection of the specific DNA target template. Mitochondrial
DNA (mtDNA) sequences were commonly used as it offers a series of advantages
over other genetic markers like cell nucleus DNA. The advantages of
mtDNA in lies in its high number of copies per cell and its genes evolve much
faster than nuclear ones. Therefore it contains more sequence variation which eases
the identi?cation of phylogenetically related species, for instance, between
cow and goat or differentiating between the different species of deer (Fajardo et al., 2007; Pereira, Carneiro, & Asch,
2010).

 

 

 

1.1.1.1        
Inhibition of Polymerase Chain Reaction

 

 

The amplification of DNA
extracted from samples by the PCR can be limited by the presence of inhibitory
compound (de Boer et al., 1995). The inhibitory compound may
be co-extracted from the sample or introduced during sample processing or
nucleic acid extraction, leading to disruption of the PCR amplification process
(Opel, Chung, & Mccord, 2009; Syarifah Nur
Syakira, 2016).
The major repercussion of inhibition of the PCR is a decreased sensitivity or
false-negative result (Schrader et al., 2012). Pharmaceutical excipients
can also be an inhibitor as some might have adsorbent properties that can trap
the DNA compromising the subsequent amplification process (Costa et al., 2015).

 

The potential mechanisms of PCR
inhibition could be binding of the inhibitor to the polymerase or interaction
of the inhibitor with the DNA. Another potential mechanism could also be
interaction 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 can
be 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 inhibitory
substance is listed in Table 1.3. Other than that, inhibitory substances may
also be accidentally added during transport, sample processing or nucleic acid
extraction.

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