Polymerase Chain Reaction (PCR)

What is PCR?

• Amplifying a particular piece of DNA (making numerous copies)

• A laboratory version of DNA replication in cells

* PCR can make billions of copies of a target sequence of DNA in a few hours

What reagents are needed for PCR?

• DNA sample (target sequence you wish to copy)

• Heat - stable DNA polymerase (TAQ polymerase)

• Nucleotides (A, G, C, T)

• Pair of primers (DNA fragments that are complementary to the start and end of the sequence you want to amplify)

• Buffer for the correct pH

Why is a heat - stable DNA polymerase (TAQ polymerase) used?

• PCR involves very high temperatures, so a heat stable DNA polymerase is used in the reaction (most DNA polymerase would denature and thus not function properly at the high temperatures of PCR)

• TAQ has optimal enzyme activity between 75 degrees Celsius and 80 degrees Celsius, and substantially reduced activity at lower temperatures

What happens to all of the reagents?

They are placed in a thin - walled tube and these tubes are then placed in the PCR thermal cycler

What are the steps of PCR?

1. Denaturation of DNA

2. Annealing (primers binding)

3. Extension

Step 1: Denaturation of DNA

• Occurs at 95 degrees Celsius, and mimics the function of DNA helicase by breaking the hydrogen bonds between the bases, separating the two strands

Step 2: Annealing (Primers Binding)

• At 40 - 65 degrees Celsius, the primers anneal (or bind to) their complementary sequences on the single strands of DNA

• Each primer is complimentary to opposite ends of the two target DNA strand

Step 3: Extension

• At 72 degrees Celsius, TAQ polymerase extends the DNA by adding nucleotides to the 3’ ends of the primer

What happens after each cycle?

• At the end of the first cycle, two DNA helicases have been formed identical the original sequence

• At the end of the second cycle, four DNA helicases have formed

• The sequence of steps (cycles) is repeated many times to create multiple copies of the one DNA sequence

How can PCR be used to treat disease?

• Primers can be created to only bind and amplify certain alleles of genes or mutations of genes

• Some diseases that can be diagnosed with PCR include: Huntington’s disease, cystic fibrosis, and HIV