PCR – Polymerase Chain Reaction

PCR (Polymerase Chain Reaction)

A technique used to make millions of copies of a small DNA segment quickly and cheaply.

Inventor of PCR

Kary B. Mullis; won the Nobel Prize in Chemistry in 1993.

Purpose of PCR

Amplify DNA when the sample is too small for testing or research.

Main Uses of PCR

Genetic testing, DNA sequencing, forensics (DNA fingerprinting), paternity tests, and detecting microbes in water.

PCR Requirements

Template DNA, primers, DNA nucleotides (dNTPs), Taq DNA polymerase, and buffer solution.

Template DNA

The DNA that contains the sequence you want to copy.

Primers

Short single-stranded DNA pieces (20-30 bases) that mark the start and end of the DNA segment to be copied.

Forward and Reverse Primers

Two primers used; one binds to each strand in opposite directions.

dNTPs (Deoxynucleotide Triphosphates)

The building blocks (A, T, G, C) used to make new DNA strands.

Taq DNA Polymerase

Heat-tolerant enzyme from Thermus aquaticus; adds new nucleotides to the growing DNA strand.

Buffer

Keeps the right pH and conditions for PCR to work properly.

Thermocycler

Machine that changes temperature automatically to carry out PCR steps.

PCR Stages

Denaturation, Annealing, Extension.

Denaturation (94-95°C)

High heat separates double-stranded DNA into single strands by breaking hydrogen bonds.

Annealing (50-65°C)

Temperature is lowered so primers can attach (bind) to single-stranded DNA templates.

Extension (72°C)

Taq polymerase adds new nucleotides to build complementary strands in the 5′→3′ direction.

Thermal Cycling

Repeating the 3 steps (20-40 times) to double the DNA each cycle.

PCR Amplification

Each cycle doubles the DNA amount: 1→2→4→8→16→32 etc.

Example Calculation

After 8 cycles, 2⁸ = 256 double-stranded DNA molecules.

Direction of DNA Synthesis

Always built 5′→3′; template is read 3′→5′.

Total Time

One full PCR reaction can be done in under an hour.

Gel Electrophoresis

Technique used after PCR to check DNA size and quantity.

DNA Movement in Gel

DNA moves toward the positive end (anode) because it is negatively charged.

Smaller DNA Fragments

Move faster and farther through the gel; give clearer results.

Larger DNA Fragments

Move slower and stay closer to the top of the gel.