PCR Fundamentals

Learn the definition of PCR as a DNA amplification technique developed by Kary Mullis in 1985. Identify the five essential components: DNA template, DNA polymerase, primers, dNTPs (nucleoside triphosphates), and buffer with salts such as magnesium.

Design short oligonucleotide primers that are complementary to the end regions of the target DNA sequence. Mix the DNA polymerase, primers, DNA template, dNTPs, salts, and buffer at pH 8.5 in a reaction tube to optimize enzyme activity.

Heat the reaction to approximately 94°C to denature the double-stranded DNA template, causing it to unwind and separate into single strands.

Lower the reaction temperature to the annealing temperature, allowing forward and reverse primers to bind specifically to complementary regions on either side of the target sequence on both DNA strands.

Raise the temperature to 72°C and allow DNA polymerase to extend the primers by adding dNTPs, creating new DNA strands complementary to the template. Repeat the heating, annealing, and extension cycle 30-40 times to achieve exponential amplification.

Perform an initial incubation at 95°C to activate the polymerase and denature target DNA, then repeat 30-40 cycles of: 30-second denaturation at 95°C, 1-minute annealing at primer-specific temperature, and 1-minute extension at 72°C. Complete with a final 5-minute extension at 72°C.

Load the amplified PCR product onto an agarose gel and perform electrophoresis to separate DNA fragments by size. Compare samples to molecular weight markers to verify correct fragment size and amplification efficiency.