Microbiology, part 42: Genetics - Gel Electrophoresis & Polymerase Chain Reaction (PCR)

Hi, I'm Cathy with Level Up RN. In this video, we're going to go over two important tools used in genetic engineering. This includes gel electrophoresis and polymerase chain reaction, or PCR. At the end of the video, I'm going to give you guys a little quiz to test your understanding of some of the key facts I'll be covering. So be sure to stay for that. And if you have our Level Up RN microbiology flashcards, be sure to pull out your flashcards so you can follow along with me.

Agarose gel electrophoresis is a technique used to separate DNA fragments by size. It is used in a variety of applications, such as forensics and paternity testing. Let's take a look at how this technique works using an illustration from our microbiology flashcard deck. First, agarose gel is poured into the casting tray with a comb in place. After the agarose polymerizes, the comb is removed, which leaves wells in the gel. DNA samples that have been stained are then placed in those wells. In one of the wells, a DNA ladder is placed, which is a solution that contains DNA fragments of known sizes. This will allow us to compare our sample DNA fragments against these fragments of known size later on.

Next, the gel plate is placed into a buffer solution and an electrical current is applied. This causes DNA molecules which are negatively charged to move from the negative end of the gel where the wells are towards the positive end of the gel. Smaller molecules are able to travel faster through the gel, whereas larger molecules will be slower. DNA fragments that have the same size will migrate the same distance through the gel matrix, forming a band in the gel. And because we stained the DNA fragments prior to using gel electrophoresis, these bands will be visible when observed with UV light. The sample bands can then be compared to bands of known size in the DNA ladder, which is shown on the left-hand side of the image. This allows us to determine the approximate size of the DNA fragments in our sample.

Next, let's talk about polymerase chain reaction, or PCR, which is a technique used to rapidly replicate a specific DNA sequence so it can be studied in greater detail. So for example, PCR could be used to replicate DNA segments, which can then be separated and visualized using gel electrophoresis. PCR is used in many applications, including genetic analysis, research, and the diagnosis of many infectious diseases. PCR uses an instrument called a thermal cycler, as well as the following ingredients: the target DNA template, DNA primers, deoxynucleotide triphosphates, which are the building blocks to make new DNA strands, as well as a heat-stable enzyme called Taq DNA polymerase. Of note, Taq DNA polymerase is extracted from the bacterium thermus aquaticus, which is a thermophilic bacterium that lives in extreme environments, such as the hot springs in Yellowstone National Park.

With PCR, the DNA sample is amplified in three main steps. This includes denaturation, annealing, and extension. During denaturation, the DNA sample is heated to approximately 95 degrees Celsius, which causes the double-stranded DNA to separate into single-stranded DNA. During annealing, the temperature is lowered to about 50 degrees Celsius, which allows DNA primers to attach or anneal to specific locations on the DNA strands. Then during extension, the temperature is raised to approximately 72 degrees Celsius, which allows Taq DNA polymerase to synthesize a complementary strand of DNA for each of the single strands. These three steps are repeated approximately 30 times. And each time the cycle is repeated, the number of DNA copies doubles. So during cycle one, we started off with one DNA molecule, and we ended up with two. Then during cycle two, we started off with those two DNA molecules and ended up with four. And during cycle three, we went from four to eight. And the number of DNA copies will continue to double with each subsequent cycle. With polymerase chain reaction, a single DNA molecule can be amplified to produce millions or billions of copies, and the entire process can be completed in just a few hours.

All right, it's quiz time, and I have four questions for you.

Question number one. When using gel electrophoresis, the blank is a solution containing DNA fragments of known sizes.

The answer is...DNA ladder.

Number two. After using gel electrophoresis, will larger or smaller DNA molecules be closer to the positive end of the gel?

The answer is...smaller fragments because they're going to move faster through the gel, so they will be closer to the positive end of the gel as compared to larger fragments.

Number three. The double-stranded DNA segment is separated into single strands during which step of PCR?

The answer is...denaturation.

And number four, if eight copies of DNA are present at the beginning of a PCR cycle, how many will be present after the cycle?

The answer is...16.

All right, that's it for this video. Hope you did great with that quiz, and I hope you found this video to be helpful. Take care, and good luck with studying.

[BLOOPERS]

Which is a technique used to rapidly-- the target DNA template, DNA primers. [No?].