Genetics: Gene Expression

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Welcome to MCAT Basics, your ultimate guide to the essential topics you need to master for the MCAT. Brought to you by the physicians at Med School Coach. Every week, Sam Smith breaks down high yield MCAT topics, ensuring you're primed for success on test day. Join SAM as we explore the most crucial subjects outlined by the AAMC, pulled from official practice materials and third party resources. Get ready to elevate your MCAT game with topics tailored to maximize your score potential.

Hello, I'm Sam Smith.

This podcast is gonna cover three main topics. The first of that is going to be different inheritance patterns that you'll see in the MCAT. The second is gonna be gene expression, which is a pretty high yield topic on the MCAT. And then the third is going to be different genetic laboratory techniques. So I'm gonna talk about fish. I'm gonna talk about the different blotting techniques. I'll talk about PCR and a few different forms of PCR that you may not be familiar with.

things like QPCR or real-time PCR. And yeah, so I hope this all helps and you get some good out of it.

So as I said, I want to get started with inheritance patterns. First, I'm going to talk about our sex link to traits. In some cases, a gene is present on the X chromosome. And so when the gene is present on the X chromosome, its inheritance pattern is going to be a little different than when it's inherited, when it's an autosomal trait. And so I'll go through those next.

These X-linked genes or X-linked traits are different than autosomal traits because these genes are present in different copy numbers in males versus females. Males have one X chromosome and females have two X chromosomes.

And so genes on the X chromosome can either be recessive or dominant. And when a gene is recessive on the X chromosome, it means that females need two copies of this recessive allele.

i.e. there are two X chromosomes, both must carry this recessive allele. Whereas for men, they only carry one X chromosome, so if they have that one recessive allele, they're going to have that phenotype. They're going to have that disease, usually, is the way you look at it.

For men, they receive their ex chromosome from their mother. You receive one of each chromosome from your mother and your father, and so for your sex chromosomes, you receive, if you're male, you receive a Y chromosome from your dad and your ex from your mom. So for sex-linked recessive disease, if your male and your mom is afflicted with some

sex-linked or excessive disease, you are automatically going to get that disease because you're inheriting her X chromosome. That's going to be your only allele. It's going to be the only allele for that gene. On the other hand, if you are a female, you're receiving two X chromosomes, one X from your dad, one X from your mom.

If your mom is afflicted with some disease and said the disease is, again, sex-linked recessive, you're not necessarily going to get that disease because your dad, you know, might have a different allele for that disease and you might be all right, you might not have a mutated allele or something like that. Sex-linked diseases tend to typically afflict males more than they do females.

Before I give you guys a quick example here, I want to make it clear that I've been using sex-linked and ex-linked kind of interchangeably. However, some diseases are not necessarily ex-linked that are sex-linked. You can have diseases that are linked to the Y chromosome. They're much more rare than you can kind of think through those problems like you would in X chromosome.

Obviously, females don't get a Y chromosome, so these diseases would only affect males. In terms of the MCAT, I think these X-linked diseases are much higher yield. To illustrate how these X-linked diseases affect men and women differently, I'm going to go through an example, phrase it like an MCAT question, and this example is red-green color blindness.

Red-green colorblindness is an excellent recessive disease. And so say you have a mother who is afflicted by this disease and you have an un-afflicted father. Now, what is the frequency of children born that are male and affected and that are female and affected?

So to solve these problems what I'll do is I'll draw out a Punnett square and then you know you can do a little simple math even in your head and figure it out. I'm not going to go through how to draw Punnett square just because I don't think having it described to you is really going to be very helpful. But you know go through, watch YouTube video if you don't understand. If you do great.

So after I've drawn up a Punnett square here, you can see you're going to have 100% of the sons that are born will have red-green colorblindness. And none of the daughters will have red-green colorblindness because they're saved from this disease by their father's ex chromosome that's functional. And it's always important to tie in different sociological theories, sociological topics into these discussions. So this is where tie in discrimination.

Obviously here is a form of discrimination by red, green, colorblindness. In the case it affects more men than women. A very significant form here of gender discrimination. All jokes aside, this is just to show that these ex-linked diseases affect men and women differently. A lot of times they're much more prevalent in men because men only receive one ex chromosome.

Another inheritance pattern is called autosomal inheritance. And so this is a little bit simpler. Again, you'll have autosomal recessive and autosomal dominant phenotypes. And so for recessive, you have to have two copies of this recessive allele. And for the dominant, you have one of these dominant alleles, then you're fine. You're not affected by that autosomal recessive disease.

In this case, it's a lot like the women in the X chromosome are getting two copies, one from your mother, one from your father. You can look back and use a Punnett square to figure out what the percentages are of children you'll have that are affected and unaffected. A lot of these questions on the MCAT are going to basically just ask you.

calculate the frequency that two specific parents are going to have the child that's affected with some autosomal recessive or autosomal dominant disease. And a lot of the times, you know, they might throw in what are the chances that it's a boy that's affected or a girl that's affected or they might do two traits in which you got to do a dihybrid cross or even a trihybrid. I think I saw in one practice test.

But anyway, so these questions are pretty easy. This is something you got to practice and get down, but once you do, it's not too bad. And real quickly, one thing that really did help me solve these problems pretty quick was to memorize the monohybrid and the dihybrid phenotypic ratios.

So a mono-hybrid cross refers to a single gene. So it's one gene that you're looking at with this mono-hybrid cross. The ratio for mating to heterozygous individuals is three to one, i.e. that is three people will have the dominant

allele and one will have two recessive alleles. So it's important in figuring out how many people are going to be affected with an autosomal recessive disease. That's one in every four people, right? Because you have three plus one, that's four. One in every four will be affected. The ratio to remember is three to one, i.e. there's going to be

three people who carry at least one domino allele, and there's going to be one individual who has two recessive alleles. For a diheward cross, which refers to two different genes, you're going to get a phenotypic ratio of nine to three to three to one. There's going to be nine individuals who carry both domino allele, so they'll carry a dominant allele A and domino allele B. Let's just say the two genes are A and B.

And then the two threes are going to correspond to the people that carry, you know, a dominant allele of gene B but no dominant allele of gene A or the reverse where you have the dominant allele of gene A but not of gene B. And then the one

is going to refer to that one individual that carries all recessive alleles. This is pretty easy. It's going to ask you, you have these two genes. The parents are both heterozygous. What is the frequency of individuals who carry all recessive alleles or have this disease?

And so, here you can say, okay, you have nine, it's gonna be one, two, you know, you add up all the numbers, that's nine, tell them 12, 13, 14, 16, it's gonna be one out of every 16 individuals carries this disease or has all recessive alleles for the two genes.

Alright, now that I've talked a little bit about these basic inheritance patterns, let's bring it a step further and talk about some of these less known inheritance patterns, things like genetic imprinting.

So genetic imprinting or genomic imprinting is an epigenetic phenomenon that causes genes to be expressed in a parent of origin, specific manner. So that is to say that you inherit from one of your parents. You directly, whatever your parents express, either one of them, you inherit that phenotype specifically.

As I talked about before, we usually have two copies of a certain gene, right? One from your mom, one from your dad. However, for genetic imprinting, one of these two copies is completely just shut off. And this happens in a parent of origin. As I said, parent of origin dependent manner.

So, this shows up as either maternal inheritance or paternal inheritance only. There's no substitute allele, which makes imprinted genes more vulnerable to the negative effects of mutations. So, you just kind of illustrate this. You'll have an affected father, then if you have an affected father,

And it's an imprinted gene. All the children will be affected. This is, of course, paternal inheritance or paternal gene imprinting. And then you can have a maternal gene imprinting where if your mother is affected, she's going to pass it along to all the children. And that's because one of these chromosomes or one of these genes is just completely silenced. And you're only expressing a certain gene from one chromosome.

Obviously, this inheritance pattern looks very similar to sex-linked traits. However, for gene imprinting or genomic imprinting, you'll see every single child affected. If mother is affected,

and the father's chromosome is silenced in all the children because of this epigenetic modification, then you'll see every single child affected. It's not just going to affect 25% of the females, 50% of the males. You're not going to just see the males inherit this genetic disorder, but then the females in the family, not that are the children. You can see every single child affected regardless if they're a male or female.

So right now your head might be spitting, you know, you might be thinking how exactly does this happen. So there are two main factors that play a role in genetic imprinting. The first is DNA methylation. So DNA methylation tends to silence genes.

So, this can happen in a parent of origin matter, especially for genetic imprinting, right? So, you know, you can inherit the set of genes from your mother or father. Your mother's genes can all be methylated, and therefore you only express the genes that you inherit from your father. The other factor that plays a role here in genetic imprinting is histone modification.

So again, histones, they are proteins that help compact the DNA. And in this case, these histones can also be modified and they're modeled by methyl groups. Again, they're methylated and this can act either way. So this can open up the chromatin, which encourages gene expression or can close off chromatin, which will actually decrease gene expression.

And this is very dependent upon where this methylation takes place. So it's important to note here that in the case of histones, the proteins, and when they are methylated, it's the actual amino acids that make up the histone protein that are methylated. In the case of DNA methylation, it's the actual DNA that's methylated.

All right, so I just wanted to touch on that topic briefly. Let's now move into a completely different topic and talk a little bit about genetic linkage. So if you recall in the last podcast, I talked a little bit about Gregor Mendel, a little bit about his experience.

experiments, he came up with the law of independent assortment. And basically, the law of independent assortment states that the inheritance of one pair of genes is independent of the inheritance of another pair. That is, if you inherit one set of genes, you are no more likely to inherit another set of genes because you inherit a set of genes. They won't affect each other.

However, genetic linkage is the main exception to this rule or law of independent assortment. So, genetic linkage is the tendency of DNA sequences that are close together on a chromosome to be inherited together during the meiosis phase of sexual reproduction.

And so what makes the genes linked is their actual physical proximity to one another. And it is measured in recombination frequency. So what is recombination frequency? What does that term mean? Well homologous recombination refers to the crossing over event that occurs in meiosis. So if you remember from the last podcast,

In myosis, prophase, one of myosis, you have crossing over where these chromosomes meet up and then they swap various portions of their chromosomes. And as you could imagine, genes that are more closely located to each other are more likely to be crossed over together to the chromosome. They're most more likely to stay together when recombination occurs.

So, recombination frequency then is the frequency in which a recombination event happens between two genes. So, it doesn't necessarily measure the frequency that two genes are crossed over together or two genes migrate, you know, to another chromosome together doing crossover. It's the frequency of a crossover event happening between two genes.

And so this number ranges from 0% to 50%. So for 50%, these e-gens are really far apart. And these are unlinked independently assorted genes. And then for 0%, these genes are literally right next to each other. And these crossover events really never happen between these two genes. So the big takeaway here is that recombination frequency measures how linked genes are.

and a very high recombination frequency of 30%, which is in the max, means that two genes are unlinked and sort independently, and then a low recombination frequency.

means that genes are linked and that recombinations never occur between those two genes because they're so close. And I really wouldn't worry too much about the kind of complex calculations involving genetic linkage and recombination and building these maps. I wouldn't worry about these calculations that you might have seen, you know, in genetics class or whatever, because I don't think I ever saw any of them on the MCAT or any of the practice materials. These problems would probably take too long and therefore they're left off.

Alright, so let's move into gene expression now, which is a very important topic. And I'm going to cover how gene expression is regulated in transcription, translation, how that works for perkaryotes and eukaryotes. And then I'll talk about DNA repair, DNA repair enzymes and how those work.

Gene expression can be regulated at a few different levels. It can be regulated at the translational level, translational level, and also a little bit during replication, actually. And so I'm mainly going to talk about gene expression controlled at the transcriptional level because that's the most common.

but I will also touch a little bit on gain expression control at the translational level as well, protein modifications and how that happens. So if you remember from basic genetics transcription is the conversion of DNA into mRNA and then translation is conversion of mRNA into protein. So let's begin with talking about transcriptional control in prokaryotes.

when you think prokaryotes, think bacteria, and so prokaryotic genes are often found in operons. And so these genes in an operon are transcribed as a group, and they all have a single promoter.

I like to think of an opera as a van full of people that are carpooling to work you know the driver is driving on his controlling all these other people you know they're all individual people. But there's only one there's a driver there's one person who's controlling where all these people are going and so you know the opera controls a certain set of jeans you know that could be five different jeans could be ten whatever but that opera controls the transcription of.

that set of genes and they're usually related genes. So for the sake of this example, let's just say it's a fanful of a family and they're all related. And so these operons contain regulatory genes and structural genes, regulatory genes code for proteins that regulate transcription or act as binding sites for other regulatory proteins and structural genes

encode for the proteins that are not regulatory. So these are the proteins that are actually functioning within this prokaryote. So you know, say an enzyme or something like that. And regulatory proteins control how much of the operon is transcribed.

And they do this by binding to the DNA itself. And there are a few types of regulatory proteins. Again, remember, these regulatory proteins are produced by regulatory genes. So there are two main types of regulatory proteins that you're going to want to know, and you're going to want to know what they bind to. So the first is called a repressor, and this binds to a piece of DNA called the operator.

And when it's bound to the operator, it reduces transcription or presses it, which makes sense. And the other type of regulatory protein you're going to want to know is called an activator. And an activator binds to part of the DNA sequence called the enhancer. And as you could probably guess,

In the activator binds, the enhancer, it increases transcription of the operon. And it does this by helping the RNA polymerase bind to the promoter. So the promoter region is another region of the DNA or of the operon to remember. And the promoter is where the RNA polymerase binds.

It's also important to note that the operon may be inducible or repressible. So an inducible operon is an operon that is always turned off and it is turned on by an inducer, which is just some kind of small molecule that binds it. And so you can think about an inducible operon is like a car sitting at a red light.

It's always off. And then your inducer molecule is a green light. That's what starts your car going. That's what gets your car on the road. And then you also have a reducible, or excuse me, you also have a repressable operon. And so this is an operon that's always on. And then it's turned off by a co-repressor, which again is some kind of small molecule that binds to it.

And this is like a car that hits a red light. So this car is going, it's on the road, it's good. Boom, boom, boom. It's hit by a cobra presser or a red light, and it slows down and must come to a stop. Everything I just talked about only applies to prokaryotes. Eukaryotes are a little more complex. I'm gonna talk about that next, but just know that operons apply to prokaryotes in our very, very rare in eukaryotic cells.

All right, so on to transcriptional control in eukaryotes. And as I was saying, this is like 10 times more complicated than bacterial regulation. I think a little less common on MCAT from what I can remember, but still important to know.

So in eukaryotes, you have transcriptional factors. And so these are proteins that bind to enhancers or silencers in the DNA and affect transcription. And so transcription factors must have a DNA binding domain. Remember, this is a defining feature of transcription factors, DNA binding domain.

take the example of chromatin remodeling. So chromatin remodeling involves the opening and closing of chromatin to allow transcriptional machinery to access the DNA. And again, we have U chromatin, which is open chromatin, and this tends to have higher levels of transcription, therefore higher levels of gene expression. And on the other hand, we have heterochromatin, which is tightly packed DNA.

which has a lot less transcription, if any at all, and less gene expression. And so things like histone, acetyltransferases, histone deacetylases, and histone methyltransferases modify histone proteins in order to

either open up the chromatin or close off the chromatin. However, these factors are not transcription factors, and that's because they don't contain a DNA binding motif. Instead, they bind two or modify histones and affect gene expression through that route instead of going and directly binding to DNA.

So remember that, very important, just because some protein or some molecule influences gene expression does not mean that it is a transcription factor. Instead, it must have a DNA binding domain.

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All right, so now back to the region of DNA that these transcription factors bind to. Again, these are called enhancers and silencers. And so enhancers increase transcription when they're bound and silencers decrease it. And the main difference in eukaryotes compared to prokaryotes in this facet is that enhancers and silencers can actually be very far away from the promoter.

and they can be either upstream or downstream, and the DNA actually forms this kind of loop, it forms this big loop back on itself, and so that the transcription factors can be bound to enhancers in their silencers, and this bend causes them to actually be in contact with the promoter.

So if you can imagine a long snake, pretend the head is the promoter region and then, you know, the tail is the enhancer and silencer. And so how do you make this, these two come in contact with each other while you take the snake's head and its tail and you bend it together and you touch them together. This is essentially what happens in order for the enhancer or the silencer to be in contact with the promoter itself. And so again, the promoter is where the RNA polymerase attaches.

and then transcribes the DNA into mRNA. And in this bend, you're going to have transcriptional factors that bring these two regions of the DNA together, and then you're also going to have mediator proteins that perform this function as well that don't directly bind to DNA, but bind one transcription factor to another and hold this bend together.

So important thing to remember about this is that just the DNA folds over on itself and these transcriptional regulators that are these distal control elements, you know, enhancers or silencers, they're far away from the promoter and must bend in order to get in proximity and exert their control of either increasing or decreasing gene expression.

Another important form of gene expression regulation comes in the form of post-transcriptional modifications. These only happen in eukaryotic cells, so don't think this happens in bacterial cells.

And there are three main post-transcriptional modifications that you're going to need to know for the MCAT. The first is intron exon splicing. The second is the three prime poly A tail. And the third one is the five prime seven-methyl guanazine cap.

So introns are part of the mRNA transcript that are removed, and exons are part of the mRNA transcript that stay in the mature transcript. And so it's kind of backwards, kind of the thing about introns you think would be in the transcript, but they're actually out. So just think about it backwards. So introns are removed, exons stay in, and this can happen in different combinations.

This is called alternative splicing. So, you know, one transcript, you might have the intron 1 removed, and in the other transcript, you won't have that piece of DNA removed in which intron 1 becomes an exon. And so by doing this, you can change the transcript and can control gene expression.

a particular example of alternative splicing is called exon skipping. In this mode, a particular exon may be excluded in MRNAs under a particular set of conditions or particular tissue. And then this exon is omitted from the mRNA and others. So that's just called exon skipping, which refers to a particular exon of interest.

So the second post-translational modification I want to talk about is the addition of a three prime poly A tail. So the poly A tail is added obviously to the three prime end of the transcript. And it is important for nuclear export translation and also stability of the mRNA. So a longer poly A tail leads to a longer degradation time.

And therefore, this transcript can float around in the cell for longer and more of it can be translated into protein. So that affects gene expression. And the last instance of post-transcriptional modification that I want to talk about is the 5 prime G cap or the 5 prime 7 methyl guanazine cap. And so again, this is at the 5 prime end of the transcript and it protects against degradation and is very important

in being able to be recognized by a translation initiation machinery. So this is what helps the ribosome recognize the mRNA transcript.

So real quickly, I'm just going to summarize the gene expression regulation that I just talked about. So prokaryotes have these things called operons, which are these groups of genes that all have a single promoter. And they are controlled by repressor proteins, which binds to the operator.

And then you have activator proteins which bind to the enhancer, and they kind of do what they sound like to do, repress or repress activators activate. And it's important to remember that these operator enhancer genes are very close to the operon, or part of the operon. And for eukaryotes, you have enhancer and silencer regions of the DNA.

that transcription factors bind to an either increase or decrease transcription. And it's important to remember that these regulatory factors can be kind of distant, distantly far away from the promoter and in order to be in close proximity, the DNA can form these loops. And in both cases for eukaryotes and prokaryotes, you have these promoter regions where the RNA polymerase binds.

So now that I've talked or briefly touched on transcription, transcriptional regulation and regulation of gene expression, let's get into DNA repair.

So there are three main types of DNA repair mechanisms that you need to know for the MCAT, or at least this is the three that I think you need to know. The first is proofreading, or proofreading, which is done by the DNA polymerase. Then there's basic decision repair, and there is mismatch repair. So I'll go over how each of these three works.

DNA proofreading basically does exactly what it sounds like it does. The DNA polymerase, which is the replication enzyme, is able to check its work as it goes along. So if it recognizes a mistake, it can then remove that mistake by removing the incorrect base that it put into the DNA, and it can then replace that base with the correct base.

Now if you remember, DNA replication occurs in the five prime to three prime direction. So in other words, the DNA polymerase travels from the five prime end of the DNA to the three prime end. However, the proofreading function of the DNA polymerase actually reads from three prime to five prime. And so each DNA polymerase has a three prime to five prime proofreading

exonucleus domain in the actual protein itself. And so this allows the enzyme to check each nucleotide during DNA synthesis and then excise or kick out the mismatched nucleotides. And again, they do that in the 3 prime to 5 prime direction.

All right, so now moving on to basic decision repair. So nature, the very prestigious journal describes basic decision repair as a process that removes and replaces damaged or misincorporated bases in one strand of the DNA that may generate mutations if left unpaired.

unrepaired. The damaged base is modified and excised to create a single strand of DNA break that is repaired to regenerate and intact helix. In other words, you have some mismatched base and that base itself is just excised out of the DNA and then you repair it. Pretty simple.

And there's actually a really large cast of enzymes that are responsible for this base excision repair. The protein or the enzyme that recognizes these mismatches are DNA glycosylases.

And then AP endonucleases cleave these sites in order to remove that base. You also have DNA polymerases that are able to add in single nucleotides. And these, of course, are a little bit different than, you know, the DNA polymerases that go the length of the DNA during replication.

And then lastly, you have DNA ligase, which might sound familiar because it's an enzyme that is used in replication. And DNA ligase catalyzes the ceiling of the gap between this new nucleotide that the DNA polymerase added.

and strand of DNA that had been mutated or the strand in which the mutated base was cut out. So it just seals that gap and creates a contiguous piece of DNA.

So the prime example of basic decision repair is when your cell is found in DNA, right? Because it's supposed to be like that. Your cell is supposed to be in RNA only, while thymine is the base that's in DNA. So just to give you brief examples, let's say you have a uracil in your DNA. Well, the first step in basic decision repair is that the uracil base is excised, leaving an A basic site or a site that has no base.

And then the second step, your AP endonuclease removes the A basic site, i.e. the backbone that was left over after that uracil base was excised. And then step three, you have DNA polymerase and DNA ligase come in. The DNA polymerase repairs that base, puts in a new one, and ligase seals a gap and boom, your DNA is repaired, protects against these mutations, you know, eventually might cause cancer.

or some other disease. All right, so on to the last DNA repair to know, and it is mismatch repair. And so mismatch repair is a system for recognizing and repairing errors in either insertions, deletions, or missing corporation of bases that usually happens during

DNA replication and recombination as well as repairing some forms of DNA damage that happen from UV rays or reactive oxidative species, et cetera. And so this repairs mismatches like, you know, when a G pairs to a T or a C to an A or any other combo that is a mismatch.

So the enzymes for this process are a little bit more complex, complicated. I'm not going to go through those. Just know that they are called mute proteins, M-U-T, mute proteins. And there's a bunch of different ones in the, you know, they form dimers, whatever trimmers. And through these kind of complex processes, do the following.

So, the first step is a mismatch is detected in newly synthesized DNA. And once that's detected, the new DNA strand is cut and the mispaired nucleotide in its neighbors are removed. So you move a big chunk of that DNA. And then the missing patch is replaced with correct nucleotides. This is done by the DNA polymerase.

And then the DNA ligase seals the gap between that base and or the bases and the backbone. So what is the biggest difference between mismatch repair and basic decision repair? I think this is pretty important to understand mismatch repair cuts out a big chunk of bases. So you cut out the mismatch and a bunch of its neighbors. And then the basic decision repair removes a single base and replaces that single base.

Other than that, they're very similar. Of course, they have different enzymes. It's important to note that mutations in the genes that code these proteins can result in cancers because then you mutate your Mute S protein in the mismatch repair system. Now you're not able to repair these other mismatches.

and your mutations start to accumulate, and eventually you can develop cancer somewhere, and cancer metastasizes, and can lead to death, et cetera, not good. All right, so now that I've covered all these different DNA repair mechanisms in detail, let me quickly recap. So the first DNA repair system is the proofreading system, and so the DNA polymerase

is able to recognize these mismatches as it goes along, if it makes a mistake, and it can remove and replace these bases, and this occurs in the opposite direction that the DNA polymerase is traveling. The next is basic decision repair. That's where you remove a single base, and then replace that base, and then you have the most common, which is the mismatch repair system. And this will actually remove a bunch of bases around your one mismatch, and then fill that back in.

All right, on to the last topic for this genetics podcast. This is the genetics laboratory techniques. And, you know, this stuff showed up a bunch on the practice material and even on the MCAT. So, well, allegedly it showed up on your MCAT. I don't think I'm supposed to talk about it, but nonetheless, let's get it going.

The first laboratory technique I want to talk about is PCR or the polymerase chain reaction. So this is a widely used technique in molecular biology to amplify a single copy or a very few number of copies of DNA in order to generate a bunch of copies of that. So this is an amplification process in order to increase the number of a specific DNA molecule.

Most people in the lab would use this if say you have a rare gene and you want to check it out You know from one cell sequence it from one cell compare that to a bunch of different cells Well, you're not gonna be able to sequence just this gene if there's only one little molecule of it You're gonna have to amplify that and then sequence so you know there's a lot of different applications, but the process is the same

for PCR in all instances. So you have an initial phase called the denaturation phase. Then you have an annealing phase and then an extending phase. In the denaturation phase, you heat the DNA up to about 95 degrees Celsius and the DNA strands separate. And then once they're separated, you lower that temperature down to 55 degrees Celsius. And this allows the primers to bind to the DNA strands. This is called the annealing phase.

And in the last phase, which is called the elongation phase, you increase the temperature to about 72 degrees Celsius, and your polymerase binds to the primers, and then it extends and adds bases along the length of that.

DNA. And again, if you think about it, these molecules are still separate, and they have those primers in there, and this just allows the polymerase to run through and add bases onto that DNA, which is single-stranded at the moment. And so it's important to note that one PCR cycle

which is these three steps I just described increases the amount of DNA you have by two, right? Because you're creating, you're breaking apart one strand and you're creating two new strands. You're doubling the amount of DNA in one cycle.

Also important to note that these temperatures are subject to change, so different enzymes are going to operate at a different temperature, so this is going to change the elongation temperature, and then you can change it at times. There's a ton of different optimization you can do within PCR.

And something important to understand for PCR and for all these techniques is what do you need in order for this reaction happen? What chemical components do you need? Well, you need number one, a DNA template. So this is what you put in that you want to be that you want amplified. And you need a DNA polymerase enzyme.

You need primers, which the DNA polymerase recognizes, and these are usually pretty short sequences, you know, maybe 20 sequences long, or 20 base pairs long, and these bind to the DNA when it's been opened up. Then, of course, you need these nucleotides, right, because you need something for your DNA polymerase to add to these open up strands, and then you need a reaction buffer. And, you know, buffer just makes sure that these enzymes work, then nothing degrades.

And so how do you choose the right primer? Well, primers are these short, all-ago nucleotide sequences, 8 to 60 base present length. And if your purpose of your project is to randomly generate sequences or randomly amplify sequences, they can be whatever. But a lot of times, you must know these sequences that you want to amplify, because you need to design primers in order to bind to these regions that you can then amplify.

You may also see the term QPCR or RTPCR, and I'll talk about those in a sec when I talk about different types of blotting, but these are for studying gene expression or quantifying gene expression.

All right, so let's talk about blotting. So what you need to know about blotting is essentially what the different blotting techniques are used for. You don't really need to understand the procedure. Why is it called blotting? I would just ignore that. Just know exactly what these things are trying to look at, what information you're trying to get from them.

So a western blot is used to detect the presence of a particular protein in a mixture. The probe that is used is therefore not DNA or not RNA, but antibodies, right? Because antibodies can bind to specific proteins. And this technique is also called immunoblodding, because your immune system uses antibodies.

So, Western blot proteins. All right, moving on. So, the Southern blot is a technique for detecting specific DNA fragments in a complex mixture. This technique was invented in the 1970s by Edward as Southern and has been applied to detect restriction fragment length polymorphisms and variable number tandem repeat polymorphisms.

And you might be saying, what the hell are those? Or you might have a good understanding with that. But just know that these can be used to identify individuals. Everybody has their own, you know, different pattern of restriction length polymorphism. Each individual has a different number of variable, variable, tandem repeats within their genes. So again, southern blotting is used to detect specific DNA fragments in a mixture.

All right, on to northern blotting. Northern blotting is used for the detection of a specific RNA fragments. And this technique is called northern simply because it's similar to the southern blotting technique. The person who invented this was not named northern. That'd be kind of a crazy coincidence.

Nonetheless, the northern blot is used to detect gene expression. And so, you know, one way to measure gene expression is through different mRNAs, and so mRNAs a form of RNA. So, northern blotting, think gene expression.

Another term you might see is RTPCR, which stands for reverse transcription PCR. So what that does is it takes mRNA and converts it to CDNA, which is complementary DNA. And then that complementary DNA can be sequenced.

And again, you can assay gene expression. You can see what genes are being expressed. You can look at differential gene expression, et cetera. Another term you're going to see is QPCR, or quantitative PCR. And this is also called a real-time PCR. And I know what you're thinking right now. And that is, why did some dumbass scientist decide it was a good idea to abbreviate

Reverse transcriptase, PCR, RT-PCR. And when there is such thing as a real time PCR.

And I fully agree with you. Makes no sense. Stupid. However, just know that RT-PCR stands for reverse transcriptase PCR, not real-time PCR. However, they kind of measure the same thing. You're looking at gene expression. They both do that. So if for some reason you can't remember that on the MCAT, probably not the biggest deal in the world. But yeah, worth mentioning.

So how exactly does QPCR help you look at gene expression? Well, QPCR basically allows you to watch a PCR reaction as it goes. So say you have some mRNA species that you want to look at.

you know, quantify it, you know, how much of this gene is there? Well, if you know its sequence and you can design primers to bind to the ends of it, then you can do a PCR reaction. So, say you have those primers, binds to your gene, your mRNA, your CDNA, and it

We'll start to amplify that. Well, the QPCR also has some kind of fluorescent dye that it's using. And so, usually this is a probe. And so, your probe binds somewhere on this DNA sequence. And as your polymerase goes across, it hits that probe and that probe fluoresces. So, over time, you're going to get more and more fluorescence and you can track that fluorescence.

And so that fluorescence then corresponds to some concentration of DNA. And so then you can use standards and you figure out what that concentration or the original concentration of your gene was. And that's how you measure the amount of gene that you have in a sample, QPCR.

But again, the most important thing to know is that for real-time PCR or QB-SAR and reverse transcription PCR, you are looking at gene expression. And the same goes for northern blotting. All right, the last molecular biology technique that applies to genetics and I want to talk about is fish or fluorescence in Z2 hybridization.

So the purpose of fish is basically to see where in a chromosome a certain sequence lies or certain sequences lie. It's a pretty simple concept really. What you do is you throw in a probe that binds to a particular region of a chromosome and that probe fluoresces and then you can literally image it and see where it's fluorescing from.

And so it's useful, for example, to help a researcher or clinician identify where a particular gene falls within someone's chromosome. The first step then is to prepare short sequences of single-stranded DNA that match a portion of the gene that the researcher is looking for. So this is like a primer, right? Because you have to know the DNA sequence before and you create that single-stranded sequence called a probe.

The next thing that the researcher would do then is attach some kind of fluorescent dye to that probe so that it can be seen, and then you mix that probe in with your DNA template or your chromosomes, and then the researcher is actually able to visibly see where on the chromosome that genetic sequence lies.

All right, so real quick recap of everything I just talked about. So PCR is used in order to amplify a certain sequence. And you can either know that sequence and design primers for it, or you can just amplify a bunch of whatever the hell you want to amplify.

the whole genome. And in that way, you don't need specific primers. And then you have these different blotting techniques, Western, which is for proteins, Southern, DNA, and then Northern RNA. And another thing that Northern is good for is measuring gene expression. And then you have

reverse transcription PCR, quantitative PCR, which is the same as real-time PCR. And these two methods are used to measure gene expression. And then lastly, you have fish or fluorescence in Z2 hybridization. And it is used in order to see where in the chromosomes specific sequences lie, specific DNA sequences lie, that is.

Alright, now for the MCAT advice of the day segment. So my advice for you today is pretty simple. And that is spend more time than you did taking practice tests and also practice materials that you spend doing them. Spend more time analyzing

You know, why you got questions wrong, why you got questions right. What is the MCAT trying to have you think? So spend more time analyzing the work that you do and actually doing the work.

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