| THEORY & FORMULAE |
The polymerase chain reaction (PCR) is a method for the amplification of a specific segment of nucleic acid. PCR has found wide applications e.g. in biological evidence in forensic cases, identifying contaminating microorganisms in food, diagnosing genetic diseases, mapping genes to specific chromosome segments. PCR is an exponential amplification process wherein the product of one cycle serves as the template in the next cycle. The products of PCR is termed amplicons and the DNA segment amplified is called the target.
The melting point of double-stranded DNA is that temperature at which half of the molecules is in double-stranded conformation and half is in single-stranded form. It is essential to know this temperature to help ensure that an adequate denaturation temperature is chosen for thermal cycling, one that will ensure optimal amplification reaction. Most PCR reactions contain salt. Potassium chloride is frequently added to to ensure the activity of the polymerase, while magnesium is a cofactor in the of the DNA polymerase enzyme. Also, DNAs having a higher G+C content will require more energy (a higher temperature) to denature them.
The formula given by Wetmur & Sninsky (1995) for estimating the melting point of long duplex DNAs is as follows:
where
Tm = melting temperature in degree centrigrade
[K] = molar concentration of monovalent salt ions
[Mg] = molar concentration of divalent salt ions
[S] = total salt concentration
FG+C = % fraction of Guanine and Cytocise bases in amplicon
L = length of amplicon in bp in amplicon
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