PCR amplification

PCR is a method used to prepare billions of copies of specific DNA sequences.

Usually to analyze DNA sequence or length, a larger number of copies of the specific DNA sequence is required than that found in a typical sample. In addition, the PCR reaction is highly specific, meaning that it will only produce copies of a desired sequence from the template (sample) DNA. This specificity is ensured by the primers, which are designed to be complementary and anneal to specific regions on each side of the DNA region of interest (target region). These features make PCR a powerful technique to be used, for instance, in genotyping of markers, before sequencing, or in various DNA tests.

Figure 1. PCR consists of three steps: 1. Denaturation, 2. Annealing, and 3. Extension. The steps are repeated many times (often 30), producing billions of DNA copies of specific regions

PCR steps

To prepare billions of DNA copies, many repeated cycles of DNA synthesis are performed in one PCR tube. Each cycle includes three distinct steps defined by the temperature (Figure 1). All cycles are performed without intervention in a PCR machine, which can change the temperature automatically after each step.

1. Denaturation step (95ºC): At this high temperature, the hydrogen bonds holding together the two DNA strands are broken, and the DNA strands fall apart. The single-stranded DNA is now available for copying.
2. Annealing step (54ºC): At 54°C, short DNA pieces called primers bind at complementary sites of the template DNA. The primers define the target sequence, which is the specific region of DNA that will be copied. Annealing temperature is calculated from the primer composition (the number of nucleotides as well as number of guanine and cytosine). Normally, you would need to calculate the optimal annealing temperature for each primer. In this case, we consider 54°C as the annealing temperature for all the primers.
3. Extension step (72ºC): At 72 ºC, an enzyme called DNA polymerase is responsible for copying DNA. It recognizes the 3′ end of a primer bound to a template strand and starts copying the template DNA.

By the end of one cycle, parts of the initial DNA strands have been doubled in number. By the end of, e.g., 30 cycles, usually performed in PCR, at least 1 billion (2³⁰) copies of the target sequence will be present in the tube. For performing PCR, you need to add a thermostable DNA polymerase, nucleotides, primers, and the DNA that you want to use as your template.

PCR reagents

Several reagents are required to perform a PCR experiment;

• Primers: Primers will bind at a specific DNA sequence and mark the beginning of the DNA amplification
• Nucleotides: Nucleotides are required to build the new DNA sequence
• Polymerase: Polymerase is an enzyme that assembles the nucleotides on the basis of the template sequence
• Template DNA: A template is required to be the basis of new DNA sequence amplification

When preparing for a PCR experiment, you must be extra careful of potential contamination. PCR is a very powerful technique to amplify DNA. This means that if you have a tiny contamination (for example, DNA coming from other samples), this DNA can also be amplified, competing with the original template and destroying your experiment's results. To prevent contamination, you need to always use gloves and work in a very clean environment (change the pipette tips when you are pipetting from a different container, tie your hair, do not cough or sneeze around the PCR workbench, and others).

As described above, error from a PCR experiment can easily skewed the result. This is why we need to minimize the error probability from PCR especially when we are continuing with another extra-sensitive technique such as Next Generation Sequencing (NGS). Typically the PCR step in NGS only consists of 10-12 cycles. The error that is created in the PCR step will show as mismatches in the alignment of NGS data, especially errors in early PCR rounds that will show up in multiple reads, falsely suggesting genetic variation in the sample.