Part 2 - PCR Amplification

Click here to learn What is PCR?

Step 1: Add Master Mix

To prepare the polymerase chain reaction (PCR), we will add the PCR Master Mix solution to our sample DNA. The PCR Master Mix solution (in the red-capped bottle) contains the following: water; a buffer to keep the mixture at the correct pH for the PCR reaction; large quantities of the four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process (What are primers?); and a heat-stable DNA polymerase that extends the copy DNA strand.

At the same time as the test reaction, we will prepare negative and positive control reactions. Instead of the sample DNA, the positive control reaction contains positive control DNA (the solution of 16S rDNA in the green-capped bottle) while the negative control reaction contains sterile deionized water. Both reactions contain the PCR Master Mix solution.

Step 2: Run PCR

Once the reaction tubes are loaded onto the thermocycler (the "PCR machine"), the automatic process of DNA replication starts (refer to the animation). The machine used in this lab has readouts that describe what is happening:

(from left to right: temperature, time remaining, cycle number, melt, anneal, and extend)

The temperature control is set up as follows:

• Initial incubation step: 95°C for 10 minutes
  
• 30 cycles of the following sequence of steps:
  • Melt: 95°C 30 seconds
  • Anneal: 60°C 30 seconds
  • Extend: 72°C 45 seconds
• Final extension step: 72°C 10 minutes
  
• Final step: 4°C store at this temperature
  

During each cycle, the first step (melt) is to separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this temperature, the primers bind (anneal) to the single-stranded DNA. (The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it's more likely for the DNA strands to bind to the primers instead of to each other.) The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.

The three steps—the separation of the strands, annealing the primer to the template, and the synthesis of new strands—take less than two minutes. Each is carried out in the same vial. At the end of a cycle, each piece of DNA in the vial has been duplicated. The cycle can be repeated 30 or more times, and each newly synthesized DNA piece acts as a new template. After 30 cycles, 1million copies of the initial DNA piece can be produced.