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Gel electrophoresis and genetic testing



Cellular and molecular biology

Gel electrophoresis and genetic testing


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High Yield Notes
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Gel electrophoresis and genetic testing

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Content Reviewers:

Rishi Desai, MD, MPH


Tanner Marshall, MS

DNA is a huge molecule consisting of a long sequence of 4 nucleotides - adenine, or A, cytosine, or C, thymine, or T and guanine, or G.

What’s more, we have 46 of these DNA molecules - compressed in our 46 chromosomes - and each of them is packed with thousands of genes that code for our various traits - like hair color, eye color, and even whether or not we have a genetic disease.

Now, sometimes genetic diseases can be caused by a mutations in a single gene - and to identify such a particular needle in the DNA haystack, we can use a tool called gel electrophoresis.

With gel electrophoresis, first, DNA is chopped up into smaller fragments using restriction enzymes - which are enzymes that break the DNA at specific nucleotide sequences. Then the DNA fragments are poured into a well within a piece of agarose gel.

The gel looks solid but it’s actually only semi-solid - and on a microscopic level it looks like catacombs filled with water.

There’s a negative charge placed at the end with the wells, and a positive charge placed at the far end of the gel.

And an electric current is passed through the gel and that pulls the negatively charged DNA fragments through the gel catacombs - towards the positive end.

The key is that the smaller fragments are more nimble and can move more quickly through the gel than the larger fragments.

Now, ideally, if we’re looking for a mutation and we had an easy way to spot it - then life would be easy! Unfortunately, finding a mutation is sometimes a bit trickier.

But this is where restriction enzymes come in so handy. Let’s say that we used a restriction enzyme called EcoRI to digest the DNA.

EcoRI binds to every G A A T T C sequence of DNA, and breaks the DNA between the G and the first A. So if we have a DNA sequence like this, then there would be three G A A T T C sites where the restriction enzyme EcoRI would break the DNA, and the resulting fragments will look like this.

If this DNA is then put into a well on a gel electrophoresis, and if a current is passed through, then the gel would result in four bands.

Each band would represent a different sized fragment of DNA, and would tell us that there were three restriction enzyme sites in the DNA.

Now, an important point to make is that restriction enzymes can cut any kind of DNA - all they need is their specific site to bind to.

However, the way each person’s DNA is cut varies amongst individuals - meaning the resulting fragments can vary in length from person to person.

A fancy word for variation is polymorphism - so we can go ahead and call it restriction fragment length polymorphisms - or RFLPs for short.

Now, if that polymorphic sequence also happens to be a site where a restriction enzyme can cut the DNA, then it’s called a polymorphic marker.

That’s because that sequence marks out a specific spot on the DNA that we can identify polymorphisms in, if we use the restriction enzyme to cut the DNA.

You see, because these enzymes break the DNA at specific nucleotide sequences, that also means that they won’t break the DNA if there’s a mutation in that specific nucleotide sequence.

For example, let’s imagine that there’s a mutation in a single nucleotide - also called a single nucleotide polymorphism - or SNP.

Now let’s say that mutation is in a restriction enzyme binding site, like this, where a single A turns into a G.

Then that would change the middle binding site for EcoRI, no longer allowing the enzyme to bind and cut at that location. In this scenario, after using the restriction enzyme EcoRI, our fragments look like this:.

So only three fragments, and the middle fragment is longer than it used to be. And in gel electrophoresis, we’ll see only three lines, which means that this DNA has a mutation. Bingo.

Another type of polymorphism is called a short tandem repeat polymorphisms, or STRPs - also called a microsatellite.

So, an STRP is a when a certain DNA motif, say a trinucleotide sequence like C A T, is repeated a lot, usually 5 to 50 times. Perhaps even more in cat lovers.

Genetic testing, also known as DNA testing, allows the genetic diagnosis of vulnerabilities to inherited diseases, and can also be used to determine a child's parentage (genetic mother and father) or in general a person's ancestry or biological relationship between people. In addition to studying chromosomes to the level of individual genes, genetic testing in a broader sense includes biochemical tests for the possible presence of genetic diseases, or mutant forms of genes associated with increased risk of developing genetic disorders. Genetic testing identifies changes in chromosomes, genes, or proteins. The variety of genetic tests has expanded throughout the years. In the past, the main genetic tests searched for abnormal chromosome numbers and mutations that lead to rare, inherited disorders. Today, tests involve analyzing multiple genes to determine the risk of developing certain more common diseases such as heart disease and cancer. The results of a genetic test can confirm or rule out a suspected genetic condition or help determine a person's chance of developing or passing on a genetic disorder. Several hundred genetic tests are currently in use, and more are being developed.