Introduction

Polymerase chain reaction (PCR) is an artificial in vivo (outside of the cell) process simulating DNA replication of a selected segment, allowing the selection and rapid amplification of a DNA fragment. The PCR machine is a thermal cycler which controls the temperature inside the container to a very accurate scale, facilitating PCR.

Reaction Mixture

Reaction mixture is the aqueous solution containing all the necessary ingredients (eg. DNA, dNTPs, Buffer, Primer and DNA Polymerase) , in performing PCR. It will be directly heated and cooled in the PCR thermal cycler, which allows for the denaturation (separation) of DNA strands, annealing of primer and extension of dNTPs.

1. DNA sample
2. Original un-selected double-stranded template DNA
3. dNTPs
4. Deoxynucleotides, which is the monomer of DNA molecules. Free-floating nitrogen bases. dNTPs bind to single-stranded DNA during DNA synthesis.
5. Taq polymerase
6. The denaturing of DNA requires a high temperature of 95℃. Normal enzymes denature at such high temperature. Taq polymerase is isolated from a heat-tolerant bacterium. To prevent the denaturing of DNA polymerase, Taq polymerase is used.
7. Primers
8. Primers are single-stranded short sequences of DNA (usually 18-22 bp) acting as the starting points for DNA synthesis. Two primers are used in each PCR reaction. They are designed so that they flank the target region (region to be amplified). The design will be discussed later.
9. Buffer
10. DNA is sensitive to pH. At low pH levels (< 7), the chemical bonds that bind nucleobases to DNA are broken by hydrolysis. At higher pH levels (> 10), deprotonation disrupts the dinucleotide bonds that form the double helix. Buffer resists pH changes by chemically neutralizing small amounts of added acidic or basic compounds, thus maintaining the overall pH of a medium.

Steps of PCR