PCR and Electrophoresis (Victoria Guidry)

PCR — which stands for Polymerase Chain Reaction — is a laboratory technique that allows researchers to multiply the number of copies of  a DNA gene region outside of the body where it can be observed. This occurs by mixing the DNA sample with free nucleotides, DNA primers, and a DNA polymerase.

The DNA polymerase used in PCR is isolated from a bacteria called Thermus aquaticus. This thermophilic bacteria has DNA polymerase that are viable at high temperatures (Taq polymerase has an optimal temperature of about 70°C). This is useful because before it can be copied via PCR, the DNA needs to be denatured — a process that involves exposing the sample to temperature that would destroy human DNA polymerase. DNA primers — short nucleotide sequences —  are added to the sample because DNA replication cannot occur without primer.

The steps to PCR involve:
1. Denaturing (94°C) — heating the sample to separate the strands of DNA
2. Annealing (50°C-65°C) — cooling the sample to attach primers to DNA strands
3. Extending (72°C) — raising the temperature to allow Taq polymerase to copy DNA

PictureFigure 1: PCR Steps

This process is usually carried out in a thermal cycle, which will repeat this process multiple times (usually 30-35 cycles).

Gel Electrophoresis allows the products of PCR to be easily seen. First, a gel will be made by mixing agarose powder and water.

The gel — which will have about 8 wells on one end– will be placed in the electrophoresis plate with the wells toward the anode (negative). The plate will be filled with buffer solution to completely cover the gel. The DNA sample will be loaded into a well. Typically, a DNA ladder is also loaded into a well. The cover is put on and the gel is ran at a specified voltage, causing the DNA particles to move from the anode through the gel toward the cathode (positive).

The DNA fragments will be separated by size, with the larger segments staying closer to the wells and the smaller segments moving further away.

image

Figure 2: Sample Gel with PCR products

PCR can be used in many fields, including genetic testing (paternity tests), forensics (sample to suspect), and diagnostics (the presence of bacterial or viral DNA).

Sources:
https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction
https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-sequencing-pcr-electrophoresis/a/polymerase-chain-reaction-pcr
Figure 1: https://prettyincrediblegirls.weebly.com/home/polymerase-chain-reaction-agarose-gel-electrophoresis-what-do-these-bands-mean
Figure 2: https://www.omicsonline.org/articles-images/2153-0777-2-101-g003.html

Advertisements

Leave a Reply

Fill in your details below or click an icon to log in:

WordPress.com Logo

You are commenting using your WordPress.com account. Log Out /  Change )

Google photo

You are commenting using your Google account. Log Out /  Change )

Twitter picture

You are commenting using your Twitter account. Log Out /  Change )

Facebook photo

You are commenting using your Facebook account. Log Out /  Change )

Connecting to %s