Polymerase Chain Reaction (PCR)

Preservation & Stabilization

Collect a DNA or RNA sample using swabs, needles, single-use tweezers or other appropriate tools. Samples may include saliva, blood, hair, bone, skin scrapings, plants, soil and other sources. Place the sample into a tube or container then add a stabilizing reagent to help preserve it. If collection happens off-site, ship the sample under appropriate conditions so it arrives ready for preparation and testing.

Nucleic Acid Extraction and Purification

Remove the sample from its container then purify nucleic acids using lysis, organic extraction and/or column-based purification. After purification add a Mastermix. Mastermix typically includes primers, DNA polymerase and nucleotides. The reaction mix is ready for thermal cycling.

Conventional PCR

Load the reaction mix into a thermal cycler to start amplification. The starting DNA/RNA is amplified into millions of copies. PCR goes through three stages::

• 1. Denaturation: Heat the reaction to at least 94 °C (201.2 °F) in a thermal cycler. Heat breaks hydrogen bonds and separates double-stranded DNA into single strands.

• 2. Annealing: Cool the reaction to about 50 to 60 °C (122 to 140 °F) so primers can bind to their target sequences on the DNA strands.

• 3. Extension: DNA polymerase extends from the primers and adds nucleotides (A, T, C, G) to build a complementary strand.

Each cycle produces new double-stranded DNA. These steps repeat for about 35 to 40 cycles, generating millions of copies.

Post PCR Clean-Up

After amplification the DNA/RNA is ready for analysis. A common method is gel electrophoresis. Load the DNA/RNA samples into one end of a gel and place it into an electrophoresis system, where an electrical current is passed through the gel. Fragments separate into bands in a ladder-like pattern based on size and charge. After staining view the gel under UV light and compare band patterns to a known control.