Gel electrophoresis and genetic testing

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.The importance of STRPs is that their repetitiveness can be bit of an issue in DNA replication.Simply put, the enzyme in charge of replication, DNA polymerase, can “slip”, and replicate a C A T trinucleotide extra times or fewer times.

This makes our allele longer or shorter, compared with the original one.And once again, if a restriction enzyme chopped up the DNA, then a fragment of DNA with more repeats on it would be longer than a fragment of DNA with fewer repeats on it.And those differences in the size of DNA fragments can be tracked using gel electrophoresis.So gel electrophoresis allows us to visualize DNA mutations and DNA repeats, so the key is to figure out how to use these as markers to track genes that are of interest.Recall that our 46 chromosomes are actually organized in 23 pairs of 2 homologous chromosomes.When gametes like the egg and sperm are forming, paired chromosomes called homologous chromosomes exchange genetic material through a process called crossing over.In crossing over, the free ends of each chromosome twist around each other and exchange parts.This is awesome because it gives us genetic variation - however, it also makes it especially hard to track genes that move from one chromosome to another.Fortunately for us, crossing over usually abides by genetic linkage - which states that the closer two genes are located on the chromosome, the less likely it will be for them to separate during crossing over.So if our marker and the mutant gene are close to one another, it’s unlikely that they will get separated from one another during a crossing over event.For example, if there’s a known point mutation or SNP that we know is near a gene or even within a gene, then if we identify individuals with the SNP using gel electrophoresis then that probably means the person has the gene of interest.Ok, so to really take advantage of gel electrophoresis, it comes down to seeing how close a marker is to the gene of interest.To do that, one approach is to identify a family with individuals that have the disease, and to draw a pedigree.After that, we can test everyone for available markers, and then we see how they correlate with the appearance of the disease.For example, let’s say that there are 4 markers - and for simplicity, let’s say that all 4 are SNPs - point mutations in the DNA at different spots.Based on this pedigree, marker 2 correlates well with the disease, in fact, it’s found in all 7 of the diseased individuals.

However, it also appears in two healthy family members.What that means is that those two individuals have inherited the SNP, but not the mutant allele.So, during crossing over, the marker and the mutant allele got separated only in 2 out of 9 divisions, which makes the marker “2” is the most appropriate in this case, as it is the closest one to the mutant allele of the four.In practice, affected pedigrees get typed for hundreds of markers across the entire genome.

Then, we select the closest, and therefore, the best marker.Alright, as a quick recap. Genetic testing uses a multitude of methods to track down, and finally identify the mutant allele, causing the disease.First, the mutant allele must be tracked down and reliably identified, through using markers, that reliably follow the mutant allele through crossover events in meiosis.

Marker candidates can be SNPs or STRPs.Next, a pedigree is made out for the affected family, and members are analyzed for the most reliable marker.The reliability of the marker gives out a great estimate of how far away it is from the disease gene - so, the closer it is,