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Seminario 11 Técnicas de Biología Molecular aplicadas a Entidades Monogénicas - Sebastián Giusti — Transcript

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0:00Hello, how are you? In today's class,
0:04we are going to analyze various
0:06molecular biology techniques used in
0:09the context of medical genetics, which
0:12aim to detect different allelic
0:14variants present in our genome. In the
0:19first part of the class, we will focus
0:22on variants of the PCR technique to
0:25understand the foundation of this
0:27method and its variations in different
0:30scenarios that may arise in the context
0:33of medical genetics. And in the second
0:36part, we will analyze techniques based
0:39on DNA sequencing. In both cases, the
0:43goal will be to understand, on one hand
0:47, the molecular foundations of each of
0:50these techniques, and also the medical
0:54and clinical context where these
0:56techniques become necessary,
0:59understanding in each case what
1:02information they provide in each of
1:05these contexts. We will begin by
1:09reviewing what you have surely seen in
1:13previous classes, which are the
1:15fundamentals of the PCR technique,
1:18which is the acronym in English for
1:21polymerase chain reaction. This
1:25technique, which was developed in the
1:2880s by the biochemist Kary Mullis, who
1:31received the Nobel Prize in Chemistry
1:35in 1993 for it. 3 is a technique that
1:40allows for the amplification of a
1:43specific DNA segment starting from a
1:46complex mixture. Hm. It is, therefore,
1:50the reproduction of a chemical reaction
1:54where what occurs is the polymerization
1:58of nucleic acids. One aspect, an
2:04advantage of this technique, is that
2:07one can design the PCR technique to
2:09amplify a specific segment of the
2:12genome, even if the starting template
2:15used is total genomic DNA. Hm. And this
2:21technique can then, despite starting
2:24from an extensive template, total
2:27genomic DNA, for example, extracted
2:30from a peripheral blood sample or other
2:33biological material, specifically
2:36amplify a single DNA segment from that
2:39sample. In this polymerase chain
2:44reaction, the elements introduced to
2:47produce it are, as we already saw, the
2:50genomic DNA extracted from a patient
2:53that constitutes the template for the
2:56polymerization reaction. The DNA
3:01polymerase enzyme used is a DNA
3:03polymerase of bacterial origin, Taq
3:06polymerase, because it is
3:08heat-resistant. And for this enzyme,
3:13which is the enzyme that will
3:16polymerize the substrates in the
3:19amplification reactions, it needs dNTPs
3:22as substrates, that is,
3:24deoxynucleotides of adenine, thymine,
3:28cytosine, and guanine. And it will also
3:32need primers, which we will briefly
3:34look at and review to understand the
3:36role they play in this reaction. For
3:41DNA polymerase to function, this enzyme
3:44requires a buffer, a stabilizing
3:46solution that keeps the pH of the
3:49reaction stable, and divalent magnesium
3:52cations, which act as a cofactor
3:57preserving the active three-dimensional
4:00structure of the DNA polymerase. Let's
4:05focus once again on dNTPs and primers
4:09as molecular requirements for DNA
4:12polymerase activity. We just mentioned
4:18that these deoxyribonucleoside
4:20triphosphates of adenine, thymine,
4:23guanine, and cytosine are the
4:25substrates that DNA polymerase will
4:28polymerize, depending on the template
4:30used during replication. And these
4:35molecules are found in large excess in
4:38the reaction so that it proceeds
4:41quickly and effectively. Let's remember
4:46that for all DNA polymerases, including
4:49Taq polymerase, which is the polymerase
4:52of bacterial origin used here, they
4:55cannot synthesize de novo. Hm. On one
5:01hand, they need a single-stranded DNA
5:04template for polymerization, and on the
5:07other hand, since they do not
5:10synthesize de novo, this means they
5:13cannot start a new strand from scratch
5:16even with a template; rather, the
5:19enzymatic activity is restricted to
5:22extending a pre-existing strand
5:24previously paired to the template. In
5:29particular, the aspect—the chemical
5:32group necessary for this polymerase
5:35polymerization reaction—is that the
5:38last nucleotide incorporated into this
5:41pre-paired strand must have a free 3 '
5:44hydroxyl end, since this hydroxyl group
5:48at the 3' position is the necessary
5:51requirement for the next nucleotide to
5:55be incorporated in the polymerization
5:58reaction, and the new nucleotide will
6:01again have this free 3 'hydroxyl end
6:04that will allow the incorporation of
6:07the subsequent nucleotide. Precisely,
6:13primers fulfill this function: being a
6:17segment of DNA. Since the primers used
6:22during the polymerization that occurs
6:25in PCR are made of DNA to be more
6:29stable, they provide this previously
6:32paired strand that will then be the
6:36site for the polymerization reaction.
6:41But that is not the only function of
6:44primers; in addition to meeting that
6:46requirement that allows the
6:48functionality of the DNA polymerases in
6:51the reaction, primers fulfill another
6:54function: determining which region is
6:57going to be amplified. That is to say,
7:01they also have the function of
7:03conferring specificity as to which DNA
7:08segment will be amplified in that
7:10reaction. This is because in each PCR
7:15reaction, at least a pair of primers is
7:19used. Hm. One of those primers binds to
7:23one of the DNA strands, and the other
7:27primer binds to the complementary
7:30strand. Hm. When these primers are
7:35annealed during the polymerization
7:37reaction, they set the limit for the
7:40new strands, and therefore, when they
7:43are used as templates themselves in the
7:46next amplification cycle, they will
7:49provide a fixed endpoint for that
7:51amplification reaction. The PCR
7:58reaction consists of a series of
8:00amplification cycles where each of
8:03these cycles has three steps. Hm. The
8:06first step involves DNA denaturation,
8:09bringing the reaction to 95 ° for a
8:12short time. This allows genomic DNA,
8:17for example, and later all
8:21amplification products as the reactions
8:25progress, to denature. That is, the
8:28double-stranded DNA separates into
8:31single strands, since the template must
8:33be single-stranded to be a substrate
8:35for amplification by DNA polymerase. In
8:41the second step, the temperature drops
8:44to between 50 and 70 °, depending on
8:47the composition of the primers, because
8:50this lower temperature allows the
8:53primers to hybridize, to bind to the
8:56DNA strands. And then in the third step
9:01, which typically occurs at 72 degrees,
9:04which is the optimal polymerization
9:07temperature for Taq polymerase,
9:09elongation occurs, the effective
9:11polymerization of those fragments. Hm.
9:16When these cycles are repeated many
9:19times, what ends up being generated is
9:22an exponential amplification. And the
9:26exponentiality is due to the fact that
9:29in each of these cycles, the amount of
9:32template of that region to be
9:34specifically amplified doubles. So, in
9:37each cycle, you have double the amount
9:40of templates you had in the previous
9:42one. Hm. Since dNTPs, Taq polymerase
9:47molecules, and primers are placed in
9:50excess inside that tube, the reaction
9:54occurs, amplifying exponentially, and
9:57after 30 cycles, for example, there
10:01will be 1 billion specifically
10:03amplified DNA molecules, making the
10:07genomic DNA we initially added a
10:10minuscule contaminant. compared to the
10:14enormous amplification product
10:16throughout these cycles. Therefore, it
10:19is considered that by the end of the
10:23PCR reaction, we have a segment of
10:27amplified DNA with billions of copies
10:31that is virtually pure. How are these
10:36amplification products visualized?
10:41Usually, a technique called nucleic
10:43acid gel electrophoresis is used.
10:46Electrophoresis involves subjecting
10:48charged molecules to an electric field
10:51so that they migrate based on their
10:53charges. As you know, nucleic acid
10:56molecules are negatively charged
10:59because the phosphate groups that
11:02contain their ribonucleotides have a
11:06net negative electrical charge and will
11:09migrate toward the positive electrode
11:13in this reaction of molecule separation
11:18. This electrophoresis does not occur
11:21in a liquid medium, but in a gel. And
11:24this gel, if we were to see it
11:27microscopically, is built by a matrix
11:30of—it's a polymer that forms a matrix
11:33of pores, a porous matrix. And so, when
11:39one subjects nucleic acid molecules to
11:42migrate in a solution through this
11:46porous matrix, what happens is that the
11:49smaller fragments can pass through this
11:54network of pores more easily than the
11:57larger fragments, which will collide a
11:59greater number of times against the
12:01walls of this gel and will be slowed
12:03down in their speed. The final effect,
12:07then, is that electrophoresis allows
12:10for the separation of DNA fragments
12:12according to their size. To visualize
12:17the DNA fragments within the gel, a
12:21molecule like ethidium bromide is used,
12:25which is a base intercalator. It is a
12:29molecule of such a size that it can fit
12:32in between two successive DNA base
12:34pairs. Hm. And one of the properties of
12:38this ethidium bromide molecule is that
12:40when it is illuminated by ultraviolet
12:42light, it emits fluorescence. So, if we
12:46introduced molecules of a dye like
12:48ethidium bromide into the gel matrix
12:51and then developed the gel with
12:53ultraviolet light, we will see that
12:56they have separated; that is, we will
12:58see fluorescent marks where there is
13:01DNA. In this photo of this gel, we
13:05observe that different samples have
13:07been loaded into each of the lanes of
13:10this gel. For example, in the first
13:13lane and the last lane, molecular
13:18weight markers were loaded, that is, a
13:21pre-made mixture of different sizes of
13:23DNA molecules that then serve to
13:26estimate the size of the molecule that
13:28was the product of the PCR reaction.
13:31Let's imagine that we ran four
13:35reactions here: 1, 2, 3, and 4. We can
13:40see that in the first three there was
13:42an amplification product and we could
13:44estimate the size of that amplification
13:47product by comparing it with the
13:49molecular weight markers, while in the
13:52last reaction there was no
13:53amplification product at all. Sometimes
13:58, these images obtained from developing
14:01the gels are often digitally inverted,
14:04showing us dark bands on a light
14:07background, as this is usually more
14:10comfortable for our eyes for
14:12visualization; but both types of
14:15visualizations are common in research
14:18papers and textbooks. I would like to
14:24tell you that there are alternatives in
14:28electrophoresis to visualize
14:30amplification products, and
14:32electrophoresis can sometimes be
14:35performed in a capillary. Hm. That is,
14:39the system is equivalent; there is an
14:42electrophoresis in the sense that an
14:45electric field is applied and there is
14:48a negative pole and a positive pole,
14:51but now the gel is it is contained
14:56within a small, thin capillary tube. Hm
14:59. But it is also a porous matrix. And
15:04when DNA molecules are separated by
15:07size, using the same principle we just
15:10saw, visualization occurs because at a
15:13specific point in this capillary there
15:16is a detector that will provide a
15:19series of fluorescence peaks as the
15:22fluorescently labeled DNA molecules
15:24pass through, either by a base
15:27intercalator or another detection
15:30method. And the result of this
15:34capillary electrophoresis is usually
15:37observed digitally in the form of an
15:39electropherogram. Hm. But, analogously,
15:44these peaks represent what would be two
15:47bands in a gel. Hm. Where the height of
15:52the peaks implies the fluorescence
15:55intensity of the bands, and what is
15:58represented on the X-axis is the
16:00separation according to size. In other
16:04words, both types of information can be
16:07obtained by gel electrophoresis or by
16:09capillary electrophoresis. In the first
16:13case, it is the visualization of the
16:15gel and the interpretation of the
16:17banding pattern that is informative,
16:19and in the second case, it is the
16:20interpretation of the corresponding
16:22electropherogram. Here we see another
16:29example where we see at the top an
16:31electropherogram corresponding to a PCR
16:35reaction where there was a single
16:38amplification product, a single
16:40amplicon. That is the technical name we
16:43give it, and that allows us to explain
16:45this single peak. On the other hand, in
16:48a second example, let's imagine that in
16:50another PCR reaction two different
16:52amplification products were generated,
16:54and this is observed as a series of two
16:56peaks. Hm. Next, having seen the
17:04foundations of the PCR technique, the
17:07reagents a basic reaction of this type
17:10entails, and the modes of visualizing
17:13PCR products, we will try to understand
17:17the uses of this technique in the
17:20context of medical genetics. That is to
17:25say, thinking that we are going to use
17:27this technique to determine the
17:29presence or absence of specific allelic
17:32variants. Something important to
17:36consider is that PCR is a
17:38hypothesis-based technique, meaning
17:41that if we are using it in a medical or
17:45clinical context, it is because we are
17:48looking for a particular allelic
17:50variant. That is, the clinical context
17:54should suggest that we are thinking of
17:57looking for allelic variants of a
17:59specific gene, and even within the same
18:02gene, that we are looking for a certain
18:05mutation in particular. Hm. Since the
18:10design of the primers in this technique
18:13requires having hypothesized about the
18:17nature of these causes of our clinical
18:20picture. Let's see an example in the
18:25context of a monogenic entity such as
18:28cystic fibrosis. We won't get into the
18:32details of this pathology, uh, but let
18:35me tell you in advance that we already
18:38know there is a symptomatology in
18:41certain boys and girls that allows us
18:43to suspect this condition, which is
18:46characterized by mucus plugging of
18:48multiple ducts. And we also know that
18:53it originates from pathogenic variants
18:56in a chloride channel gene called CFTR,
19:02and that the most frequent mutation in
19:05the population, uh, that causes this
19:09pathology, is a mutation called
19:12phenylalanine 508, where a deletion of
19:16three nucleotides in the gene, in an
19:19exon, in a strictly coding region,
19:22correlates to the deletion of a codon
19:26that codes for the amino acid.
19:30phenylalanine, and that this absence of
19:32this amino acid has negative
19:34consequences for protein function. So,
19:39if there is a patient who has clinical
19:45signs compatible with cystic fibrosis,
19:48it could be useful, since it is known
19:51that in many patients, uh, this
19:54particular allelic variant that
19:56involves the deletion of the
19:59phenylalanine is the one present, for
20:02one to go and look if this
20:05phenylalanine deletion is present or
20:08absent at this specific position of the
20:11CFTR gene. So, in this context, I would
20:17like to show you how conventional PCR
20:20can be very useful for detecting
20:23insertions or deletions as in this case
20:26and guided by a clinical hypothesis.
20:32For that, since we know which gene we
20:37want to explore to see if it contains
20:40this insertion or deletion, we can
20:43design a pair of primers, because we
20:46know the human genome sequence and can
20:49then access it via databases and use it
20:53to, uh, design our primers; and we will
20:56design them in a region flanking the
20:59position where the deletion may or may
21:02not be. Here in this example, we are,
21:06uh, sketching at the top an allelic
21:10variant that does not contain the
21:13deletion in question and another that
21:16does. Note that since the primers are
21:20in a flanking region, in both cases, in
21:22both allelic variants, the primers can
21:25bind and will be able to generate
21:27amplicons, that is, amplification
21:29reactions in both cases. Hm. But the
21:34amplification products will be slightly
21:36different, since if we compare them
21:38with each other, the amplification
21:40product of the variant that does not
21:42contain the deletion will have three
21:45nucleotides more than the variant that
21:47does possess the deletion. Although
21:51this difference of three nucleotides is
21:54small, polyacrylamide gels, which are
21:59different from agarose gels, can be
22:01used to discriminate these differences.
22:04Hm. That is to say, in a polyacrylamide
22:08gel matrix, electrophoresis, which will
22:10separate the molecules according to
22:12their size, can occur in the same way.
22:16And let's analyze then Uh, the result
22:21of this PCR, imagining that we obtained
22:24biological samples, perhaps through
22:26blood, peripheral blood samples, from
22:29members of a family where some of them
22:32showed clinical signs of cystic
22:35fibrosis and we wanted to explore the
22:38presence or absence of this particular
22:41deletion in the CFTR gene. Let's focus,
22:46for example, on individual number four.
22:52Hm. Individual number four has a single
22:57amplification product. Hm. And if we
23:01compare it with individual number two,
23:04which also has a single, uh, amplicon
23:06fragment, an amplification product, if
23:08we compare them with each other, we can
23:11say that the amplification product of
23:13individual four is larger in base pairs
23:16than the amplification product of two;
23:18because, as we had said, the smaller
23:20fragments migrate further in the gel,
23:23they can pass through the porous matrix
23:26with less difficulty than larger
23:28fragments. So, considering, we would
23:33now have to make considerations
23:35regarding the structure of the human
23:37genome to know what has happened there.
23:42We have to consider, for example, the
23:44diploid nature of our genome, that we
23:46have two allelic variants, one of
23:48maternal origin and another of paternal
23:50origin for each of the genes. If we
23:53have, in both individual two and
23:55individual four, a single amplification
23:57product, we will say that the maternal
24:01and paternal variants are behaving in
24:03the same way, they are generating the
24:05same type of amplification product. Hm.
24:07Now then, how to understand a larger
24:11amplification product versus a smaller
24:15one? In the context of this PCR, where
24:18we use primers to amplify a certain
24:20segment of the CFTR gene, one could say
24:23that individual 4, since it has the
24:28larger sizes, its two allelic variants,
24:30the maternal and the paternal, were
24:32allelic variants without the
24:34phenylalanine deletion. 508, while
24:37individual 2 has both of its allelic
24:41variants with the deletion. That is why
24:44we see a single amplification product
24:46resulting from those two allelic
24:50variants that were used as a template
24:52during the PCR, but which generated the
24:54same amplification product. In contrast
24:58to these cases, individuals 1 and 3
25:01have two amplification products, one
25:04larger and one smaller. Hm. And so we
25:08can think that in those individuals,
25:10one of the allelic variants has the
25:13deletion and the other does not. Hm. In
25:15other words, the deletion would be in
25:18heterozygosis in these two individuals.
25:25With what we have seen so far, we could
25:28think that since insertions and
25:30deletions can generate amplicons, that
25:33is, amplification products of different
25:36sizes that will later be separated by
25:39gel electrophoresis, we can understand
25:42why PCR can be used to detect
25:45insertions and deletions. Now, what
25:48would happen if we want to detect other
25:51types of allelic variants? For example,
25:56those caused by a substitution mutation
25:59where there will be no size differences
26:03between the amplification products. To
26:09detect allelic variants caused by
26:12substitution mutations, you must use
26:16variations of the PCR technique
26:19specifically designed to detect these
26:22substitutions. And one of these
26:27variations is allele-specific PCR,
26:30which is specifically designed for this
26:34purpose. What is the design feature
26:39that allows this? Imagine we have two
26:42allelic variants that differ by a point
26:46mutation, meaning that in the same
26:49region where one allelic variant has an
26:52AT pair, another allelic variant at
26:56that same position of the same gene has
26:59a CG base pair. The key here is to
27:05design a pair of primers where one of
27:09them binds specifically at its 3'
27:11hydroxyl end, which must necessarily be
27:15base-paired to the template for
27:17amplification to occur. We have to
27:22design that primer to be
27:24allele-specific as well. In other words
27:28, if we design it so that it can only
27:30bind to one of the allelic variants,
27:32there will be a mismatch at this point
27:34in the other allelic variant. And this
27:38implies that even if the other primer
27:41is bound, one of the allelic variants
27:43will not be able to amplify during the
27:46PCR reaction, while the other will. Hmm
27:49. So, depending on whether there is an
27:53amplification product or not, one could
27:56infer which of the two allelic variants
27:58was present. For example, if we take
28:05samples from five patients to determine
28:08which of the two allelic variants they
28:11possessed and we have these results,
28:13here we see a lane as a result of the
28:16gel electrophoresis revealing a
28:18molecular weight marker; but the PCR of
28:21patient 1, and of patient 2 and 5, give
28:24us an amplification product. We can say
28:29that they had at least one CG-type
28:31allelic variant in their genome. Now,
28:36what we can also say is that
28:38individuals 3 and 4 apparently did not
28:40have any CG variant in their genome.
28:44Usually, these allele-specific PCRs
28:47need to perform another PCR to detect
28:50the other variant to have complete
28:53information. Hmm. For example, if we
28:58use primers with the same patients '
29:00genomic DNA to detect the other variant
29:03, that is, primers that can bind when
29:06the AT dinucleotide is present, but not
29:09the CG dinucleotide, we complete the
29:12information here. For instance, we see
29:16that individuals 3 and 4, which
29:17previously showed no amplification
29:19signal, now show one. Then we can say
29:23that the two allelic variants, both
29:25maternal and paternal in origin, of
29:28individuals 3 and 4 had an AT allelic
29:30variant and did not have any CG. But
29:35what can we say, for example, about
29:38individual 1, who has an amplification
29:41product in both one PCR and the other?
29:45In this case, this individual is
29:47probably heterozygous, meaning they
29:49have one allelic variant of parental
29:52origin of the CG type and another AT
29:54variant. Therefore, there was an
29:56amplification product in both cases. Hm
29:59. I leave it to you to think about what
30:03the genetic configuration will be in
30:04terms of these variants for individual
30:075, who presents amplification here and
30:11not here. Let me tell you that there is
30:18also another variant of the PCR
30:21technique called PCR-RFLP which is used
30:28to detect allelic variants with point
30:32substitution mutations, that is, that
30:35differ from one another by a
30:38substitution mutation. This acronym
30:44RFLP stands for restriction fragment
30:50length polymorphisms. And this refers
30:55to the use in the research context of
30:58endonucleases, called restriction
31:01enzymes, which are bacterial
31:03endonucleases that can cleave DNA at
31:06specific sequences. In this case, if
31:11one wants to differentiate two allelic
31:13variants, for example, let's see that
31:15this variant differs at this point. Hm.
31:18Here there is AT and here there is TA.
31:22That is to say, there is a substitution
31:24at a particular point. Whoever designs
31:29this technique has to infer if in the
31:32context where the variation one wants
31:34to detect is located, it contains any
31:37recognition sequence for a restriction
31:40enzyme. If one goes to the databases
31:44and compares the recognition sequences
31:47of restriction enzymes and finds that
31:50this variation affects one of these
31:52sites, one could say the following. For
31:56example, in this case there is a
31:57restriction enzyme which is D1 that can
32:02cut, that is, it has a recognition site
32:06in the allelic variant that had AT at
32:09this position, but when that nucleotide
32:12is substituted by its inverse TA, the
32:15restriction enzyme can no longer cut.
32:21Realizing this aspect allows us to
32:24design a PCR in combination with a
32:26cleavage reaction using this
32:28restriction enzyme, which will
32:30eventually allow us—and this is our
32:32goal—to distinguish between these
32:34allelic variants. In particular, to
32:38determine which allelic variants an
32:40individual has, um, that we are
32:44analyzing. For that, we simply design
32:49primers. that flank the restriction
32:52site where the variant we are looking
32:54for is located. And since these primers
32:57do not affect or bind to the
32:59potentially polymorphic region, they
33:02bind to both one allelic variant and
33:05the other. When amplifying by PCR, in
33:09both cases, we will obtain
33:11amplification products of the same size
33:15. And as a second characteristic step
33:18of this technique, we treat the PCR
33:20amplification products with the
33:22restriction enzyme. So, let's imagine
33:27what happens in the first step. The two
33:32allelic variants are amplified by PCR.
33:35We have millions and millions of copies
33:37of these amplification products in each
33:40PCR tube, which are equal in size. Now,
33:43when we treat them with the restriction
33:47enzyme, only one of the fragments can
33:50be cut and the other will remain uncut
33:53because the allelic variant precisely
33:56affected this enzyme's restriction site
33:59. And so now we have a polymorphism in
34:02the length of the fragments. Hm. Hence
34:06the name of the technique, and we know
34:09that DNA fragments of different sizes
34:11can be separated using the
34:13electrophoresis technique. Let's
34:18interpret once again five patients
34:20whose genetic makeup we have explored
34:23thanks to this technique. Let us recall
34:28, then, that the allelic variant we
34:30call AT here was the one that retained
34:33the restriction site and could be
34:35cleaved, generating these two fragments
34:38, while the TA variant was not going to
34:40be a substrate for this restriction
34:43reaction and would generate a longer
34:45fragment. For example, if we think of
34:50patient number four, who has a single
34:52large-sized amplification product, Hm.
34:56because it is the one that migrates the
34:57least. We can say that individual four
35:00had a maternal and paternal allelic
35:02variant with the TA-type variant, since
35:05the largest fragments are the ones that
35:08are not substrates for the restriction
35:10enzyme. Hm. On the other hand, if we
35:15think of individual one, where we have
35:18two restriction fragments and both are
35:21smaller in size than the one we had
35:23found in individual four, we can think
35:26that this individual only had AT-type
35:29allelic variants that generated these
35:32two fragments that are different from
35:35each other, but in both cases smaller
35:38than the other allelic variant. Hm. And
35:43the most complex case to interpret is
35:45found in individuals 3 and 5, where we
35:48see three amplification products on the
35:51gel. And in this case, we interpret
35:56that these individuals had a single TA
35:58allelic variant, which was not cut by
36:01the restriction enzyme and generated
36:03the larger fragment. And the other
36:07allelic variant was of the AT type,
36:09which was fragmented and generated the
36:12smaller fragments. I would now like to
36:20tell you about another application of
36:22PCR, which is multiplex PCR, in a very
36:27particular context: the context of
36:29forensic genetics, where, in this case,
36:32it is not a pathological context, but
36:35rather an attempt to determine genetic
36:37variants that allow for the
36:39establishment of biological
36:41relationships between individuals, for
36:44example, filiation relationships. Hm.
36:48And in this context, usually what is
36:52explored are highly polymorphic regions
36:56of our genome, which are short tandem
36:59repeats, or STRs, usually represented
37:03by elements called microsatellites that
37:07we analyzed in seminar number nine.
37:13These, uh, these microsatellites, these
37:17short tandem repeats, behave like
37:19allelic variants, that is to say, all
37:22human beings at the same position of
37:25certain chromosomes possess a variant
37:29of maternal origin and a variant of
37:31paternal origin of these repetitive
37:34regions. And since they are highly
37:38polymorphic, they differ from each
37:40other very frequently in the exact
37:42number of repeats that each one
37:44possesses. Hm. And since each one of us
37:50received these variants, one of
37:52maternal origin and one of paternal
37:54origin, if one explores, for example,
37:57for one, uh, for one of these
37:59microsatellites in particular, we
38:01should probably find one of those
38:04allelic variants in our biological
38:06mother and the other allelic variant we
38:09should find in our biological father.
38:15To increase the fidelity of these
38:18analyses, for example, of filiation, a
38:21single microsatellite is not analyzed,
38:25but rather, uh, in the year 1997, the
38:30FBI standardized a procedure that
38:35analyzes 13 sequences, uh, of mini
38:40microsatellites scattered throughout
38:43different human chromosomes. Therefore,
38:47the inferences will not only be based
38:50on a single microsatellite, but on
38:52multiple, uh, loci, multiple
38:54chromosomal positions that are on
38:56different chromosomes. These names that
39:01we see here, TPOX, D8S 1179, VWA, etc.,
39:05are the specific names of tandem repeat
39:12regions, which are highly polymorphic
39:15in the human population, but which all
39:18humans have at those positions on those
39:21chromosomes. So, I wanted to tell you
39:28that this multiplex PCR technique is
39:31applied to analyze the length of these
39:35regions, to determine the number of
39:39repetitions that each of these
39:42microsatellites has in a single
39:45reaction at the same time. What is the
39:51key? What specific aspect of multiplex
39:56PCR, which, unlike classical PCRs where
39:59we used a pair of primers to amplify
40:02those regions of the genome that we
40:05wanted to amplify. In this case, we are
40:10going to use many different pairs of
40:13primers, each one of them designed to
40:15amplify one of these microsatellites in
40:18particular, that is, the flanking
40:21region that allows amplifying that
40:23microsatellite which is on each of the
40:26chromosomes we analyze. Precisely
40:31because this reaction is complex,
40:34because amplicons from different genes,
40:37from different pairs of primers, are
40:40going to be mixed in the same tube,
40:43fluorophores are usually used to
40:45visualize the amplification products,
40:49molecules linked to the primers, that
40:52will emit fluorescence in different
40:55colors, thus allowing the different PCR
40:58products to be differentiated by color.
41:03Since this reaction is very complex,
41:05because we have at least 13 different
41:08amplification products, what is done is
41:11to use the same fluorophores for some
41:14genes that have amplicons of different
41:17sizes. So that by combining these size
41:21differences with differences in
41:23fluorophores, we can obtain the total
41:25information. Usually, this multiplex
41:30PCR is combined with capillary
41:32electrophoresis to then detect the
41:35different products as we saw. And in
41:40the detector, the fluorescences coming
41:42from the different colors that we used
41:45in the fluorophores of the different
41:47pairs of primers can be detected. I
41:51show you an example here in this
41:54electropherogram. As a result of having
41:59used multiple pairs of primers with
42:01different fluorophores, we obtain a
42:04series of peaks with different colors.
42:09Let's remember that on the vertical
42:11axis, the height of the peaks implies
42:13the fluorescence intensity of each of
42:15those amplification products, and on
42:17the horizontal axis, these peaks are
42:20separated based on the size of the
42:22amplicon. Hm. Digitally, one can
42:26separate these signals of different
42:27colors to perform a more detailed
42:29analysis. For example, here we see how
42:33we separate the channel of those
42:35amplification products that had a blue
42:38fluorophore in the upper part and in
42:40the lower part the channel of those
42:43green fluorophores. Note that because
42:49of how the primers were designed, one
42:50can know in which region we will obtain
42:55the amplification products of a
42:56particular microsatellite. For example,
42:59for this particular microsatellite we
43:01have two amplification products. We see
43:03two peaks. In this case, for this
43:06particular microsatellite, this
43:08individual we analyzed had a
43:10microsatellite with 12 repetitions and
43:12another with 13 of this one in
43:15particular. Conversely, for this other
43:18microsatellite from the same individual
43:21, we have a single peak. What does this
43:23mean? That the maternal and paternal
43:26allelic variants had the same number of
43:28repeats. Hm. So, by combining the
43:31information from these multiple primer
43:34pairs associated with different
43:36fluorophores, one can, uh, know the
43:41exact number of repeats an individual
43:44has for each of these tandem repeat
43:47regions and compare it, then, with
43:51potential, uh, likely subjects, uh,
43:54biological fathers or mothers, and
43:58determine the probability of whether or
44:01not a biological affiliation exists
44:04between them. Finally, I would like to
44:12tell you about this last variant of the
44:15PCR technique we are going to analyze
44:19today, which is TP-PCR, or Triplet
44:22Primed PCR, used in the genetic
44:24diagnosis of triplet repeat expansion
44:28diseases. This is a set of
44:31non-classical monogenic diseases that
44:34will be analyzed in depth. in an
44:37upcoming seminar, but they are
44:40characterized by the expansion of a
44:42three-nucleotide repeat within the
44:45affected gene. So, in the population,
44:49there are allelic variants that have a
44:52normal range of repeats, meaning they
44:55are non-pathogenic. And these, for
44:59example, in this particular case,
45:01unaffected individuals have between 5
45:03and 50 repeats of this trinucleotide in
45:05their maternal and/or paternal allelic
45:08variant. A characteristic of this
45:13disease is that some individuals have
45:15an allelic variant called a premutation
45:18with a higher number of repeats. And
45:21the premutation is characterized by the
45:24fact that those who carry this
45:27premutation do not show signs and
45:29symptoms of the pathological entity,
45:32but there is a high probability that in
45:35their gametes, an expansion of the
45:37mutation occurs, h, of a higher range
45:40that will then be associated, in the
45:43next generation, with the appearance of
45:46signs and symptoms. As you will see,
45:50this technique, what it tries to
45:52determine in the context where there is
45:55a hypothesis that there is a triplet
45:57expansion disease behind the signs and
46:00symptoms of some family members, is to
46:02determine if an individual is a carrier
46:05of allelic variants with expansions
46:07within the normal range, within the
46:09premutation range, or the pathogenic
46:12mutation range. And you might ask
46:16yourselves, why couldn't conventional
46:18PCR discriminate this? Since we have
46:21seen that insertions, for example, that
46:23variations in length could be detected
46:25by the PCR technique. But although from
46:31a theoretical point of view that is
46:33true, there are specific problems that
46:36arise for conventional PCR when it
46:39comes to detecting, uh, triplet
46:41expansions. In this case, we see the
46:46FMR1 gene as an example, which is one
46:50that can have this huge triplet
46:53expansion in its 5-prime UTR region. We
47:01mentioned that the limitation of
47:04conventional PCR when detecting
47:07amplification products of these
47:09expanded alleles is due to the fact
47:12that these regions containing multiple
47:15repeats are often a difficult context
47:18for the DNA polymerase used in these
47:21reactions to effectively amplify the
47:24entire fragment. And this causes a
47:29series of errors, a series of
47:31incomplete fragments of the potentially
47:33expanded fragment to be amplified,
47:36which results in the template not being
47:39able to be effectively duplicated from
47:41cycle to cycle, and towards the end of
47:44the PCR, we observe no amplification
47:47product if there was an expanded allele
47:50in the template. Simply put, the PCR
47:54fails and fails to amplify. When we
47:59only use a pair of primers flanking the
48:02potentially expanded region, it fails
48:05to amplify that fragment. Hm. So, if a
48:10diploid individual has an allelic
48:12variant from a parental origin without
48:15amplification, this will be amplified.
48:18But if it has an expanded allele as the
48:21other allelic variant, there will be no
48:24amplification product, and since only
48:27the amplification product of the
48:29non-expanded variant will be seen on
48:32the gel, there is a masked result.
48:36These problems were originally solved
48:39with a different technique, which was
48:41the Southern blot technique, which we
48:43will not cover here because it is a
48:46technique that is falling into disuse,
48:48but variations of the PCR technique
48:50allowed the problem to be solved. To
48:54avoid the problems of lack of
48:57amplification that occur with triplet
49:00expansion when we try to determine it
49:02with conventional PCR, this variant of
49:05PCR uses three primers. One of them,
49:10acting as a forward primer, that is, on
49:13one of the strands, is placed in the
49:15flanking region of the amplification.
49:19Then a second primer is used that will
49:25be complementary to the repeat region
49:27and a third primer that will function
49:30as a reverse primer, that is, in the
49:32opposite direction, which will bind to
49:35the complementary strand. Let us
49:39consider that the use of these three
49:41primers, in particular, the primer that
49:44binds to the repeat regions, has the
49:47following peculiarity. This primer can
49:51bind anywhere in the repeat region,
49:54generating diverse amplification
49:57products when it creates amplicons in
50:00combination with the primer facing in
50:03the opposite direction. So, let's think
50:08that in the PCR reaction we have
50:12millions of molecules acting as
50:14templates and many reactions occurring
50:17simultaneously. Consequently, one would
50:21expect from this configuration that,
50:24for example, amplification products are
50:27generated, uh, of different lengths, as
50:33we were just saying, when the repeat
50:35primer acts, and some of full length
50:37when both flanking primers act.
50:41Consequently, uh, this technique has
50:44the advantage that it will allow for
50:47estimating the presence or absence of
50:50these expanded allelic variants, uh, by
50:54two methodologies. Hm. On one hand, uh,
50:58we will try to see if there is
51:00amplification of the full expansion
51:03product, but we will have, uh, as an
51:06aid in this technique, the presence of
51:09a series of fragments of different
51:12sizes that span the different regions
51:15of the repeat. TP-PCR results are
51:20usually analyzed by capillary
51:23electrophoresis. Let us remember that,
51:28uh, these capillary electrophoresis
51:30results are usually visualized in these
51:33graphs called electropherograms, where
51:36on the vertical axis we observe that
51:39the height of the peaks, uh, reflects
51:41the level of signal fluorescence
51:44intensity, uh, the presence of
51:46amplification products and their
51:48intensity. And on the horizontal axis,
51:52what we see is the variation in the
51:55sizes of those amplification products.
51:58In this case, uh, we are observing
52:01different length ranges of those
52:03products that allow us to classify
52:06these allelic variants into the normal
52:10zone, premutation, or fully expanded
52:13mutation. Let us analyze how the
52:17results look in the case of an allelic
52:19variant that possesses trinucleotide
52:22repeats in the normal range. Here we
52:25see, on one hand, an intense
52:27amplification product of the allelic
52:30variant in the normal range, which is
52:33the amplification product of the two
52:35pairs of primers that are in the
52:38flanking regions. Hm. And we also see a
52:42series of peaks in the form of a ladder
52:44, which are those produced by the
52:46amplification products where the primer
52:49acts that can bind to the repeat region
52:51and, therefore, can yield different
52:53amplification products. But this entire
52:58ladder of peaks is also restricted to
53:00the normal range because the range of
53:03repeats that these alleles possess is
53:05relatively limited. How is it observed?
53:10the the result of the electropherogram
53:14when there is an individual who
53:16possesses a premutated variant, that is
53:18, with an intermediate range of triplet
53:21expansion. In the first place, we see
53:24that the series of peaks, uh, that is
53:27produced as a consequence of the
53:29different fragments amplified from the
53:32primer that binds to the repeat region
53:34is wider. Yes, we have a larger range
53:38of amplification of those products and,
53:41in addition, we see a stronger signal,
53:44uh, of the larger amplification product
53:46, the one produced by those primers
53:49that flank the repeat region, which is
53:52centered at a higher molecular weight.
53:56Hm. Here we don't see a single peak
53:59because usually, the longer the
54:01fragment to be proliferated, the
54:03greater the number of errors that occur
54:06in the PCR products. Hm. We also see
54:11that the peak height has decreased
54:13compared to the previous time,
54:14precisely because that amplification
54:16product is less efficient and the total
54:18amount of product we will obtain will
54:20be smaller, and its fluorescence
54:21intensity, the peak height, will also
54:23be lower. Finally, let's compare this
54:28with a carrier of an allelic variant
54:31with full expansion, where we see that
54:34the larger the expanded region, the
54:37region of peaks—hm—produced as a
54:40product of the amplification of the
54:42primer that bound to the repeat region
54:45covers a much wider range of molecular
54:48weights, and we see that the peak
54:52corresponding to the amplification of
54:55the full region has a lower height.
54:58This means that the efficiency of its
55:00production is lower. It was produced in
55:03a smaller quantity, but the size of
55:06that total product is larger than
55:09before and is in the full mutation
55:12region. In summary, this TP-PCR
55:15technique is a specific adaptation of
55:19the PCR technique to detect allelic
55:22variants due to triplet expansion. In
55:28this second part of the lecture, we are
55:30going to study the techniques based on
55:33DNA sequencing that are used in the
55:35context of medical genetics. This set
55:39of techniques focuses on determining
55:42the order of base pairs that are
55:45present in a given DNA fragment. And
55:48within this set of methodologies, we
55:51will analyze, first, the sequencing
55:54method developed by Frederik Sanger,
55:57who won the Nobel Prize in Chemistry in
56:001980 thanks to this development, and
56:03which, as we will see, was the
56:05methodology used to produce the first
56:08sequencing of the human genome in the
56:11context of the Human Genome Project.
56:16Then we will address subsequent
56:18developments from the last 15 to 20
56:20years that, based on Sanger sequencing
56:22methods, allowed it to be expanded and
56:25are collectively called high-throughput
56:27sequencing methodologies, also known as
56:30next-generation sequencing or
56:32high-throughput sequencing. To
56:36understand the Sanger sequencing
56:39methodology, we have to re-analyze some
56:44molecular mechanisms that are involved
56:46in the polymerization of nucleic acids,
56:49since it is the particular biochemistry
56:52of polymerization that Sanger used as
56:55the foundation for his technique. Let's
57:00remember again that during the nucleic
57:03acid polymerization process, DNA
57:06polymerase uses a single-stranded DNA
57:09strand as a template, but it cannot
57:12polymerize de novo; it can only extend
57:15a previously paired strand that has a
57:18free three-prime hydroxyl end. We see
57:24on the right side of the slide this
57:27three-prime hydroxyl end in greater
57:30molecular detail. We are seeing that
57:33the last nucleotide added to a chain at
57:36the three-prime position of the sugar
57:39has a hydroxyl group that will be used
57:42to form a new covalent bond with the
57:44incoming nucleotide, forming a
57:47phosphodiester bond and allowing the
57:49incorporation of the new nucleotide.
57:54Then, the one that will function as the
57:56next new terminal nucleotide of the
57:59chain will in turn have another
58:01three-prime hydroxyl end that will
58:03function in a similar way, that is,
58:06allowing the incorporation of the
58:08subsequent nucleotide. Taking this into
58:13account, Sanger devised using variants
58:18of nucleotides that would prevent the
58:20development of polymerization. Here we
58:25see a standard nucleotide that has the
58:29hydroxyl group associated with the 3-
58:32prime carbon of the sugar and a variant
58:35that was what he used, called
58:38dideoxyribonucleotide triphosphates,
58:41which lack this hydroxyl group at the 3
58:45-prime position. If one, in an in vitro
58:50polymerization reaction, adds these
58:52dideoxynucleotides, that is,
58:54nucleotides that lack this 3-prime
58:57hydroxyl end, once they are
58:59incorporated, they terminate
59:01polymerization because no nucleotide
59:03can be incorporated by binding to them,
59:06since they lack this functional group
59:09that allowed the incorporation of the
59:12new nucleotide. How then did this
59:16particular biochemistry allow Sanger to
59:19devise a methodology for the sequencing
59:21of nucleic acids? Let's think about a
59:27polymerization reaction that occurs in
59:31a laboratory tube. In it, the
59:36substrates for polymerization are
59:39introduced: a DNA polymerase, the dNTPs
59:42, that is, deoxyribonucleotides of
59:45adenine, guanine, cytosine, and thymine
59:49, and small amounts of
59:51dideoxynucleotides of the four classes.
59:55Hm. Furthermore, let's think that these
1:00:00dideoxynucleotides, which are in much
1:00:02smaller amounts than the rest of the
1:00:04natural nucleotides, will each be
1:00:06labeled with a different fluorophore.
1:00:11On the other hand, the template is
1:00:14added, which will be a DNA fragment
1:00:16whose base sequence one wants to
1:00:18determine. You might think that
1:00:23information about the sequence of this
1:00:27fragment will be necessary to design a
1:00:30primer needed to introduce as a
1:00:32condition of possibility for the DNA
1:00:35polymerase to function. A biochemical
1:00:42trick that was used in this context was
1:00:45to chemically add to the end of the DNA
1:00:48molecule that one wanted to sequence,
1:00:52that is, whose nucleotide sequence was
1:00:55still unknown, a DNA fragment that we
1:00:58are representing here with this green
1:01:01line of known sequence. When this known
1:01:06sequence fragment is chemically
1:01:08attached, one will be able to design a
1:01:11complementary primer to this known
1:01:13sequence, which will then allow it to
1:01:15act as an initiator for the
1:01:17polymerization of the region that is
1:01:19still unknown. I am not representing it
1:01:23here, but let's imagine then that a
1:01:25primer complementary to this known
1:01:27region, designed by the researcher, is
1:01:30also added to this polymerization
1:01:32reaction. Let's think about this
1:01:36reaction where, although we are
1:01:39representing the strand as
1:01:41single-stranded, it is originally
1:01:44double-stranded, but we will only use a
1:01:48single primer from the PCR that binds
1:01:52to the strand to be used as a template.
1:01:55Hm. Let's imagine as a second step, to
1:02:01make the representation of what is
1:02:03happening here more complex, that this
1:02:06reaction is occurring millions of times
1:02:08inside this tube in parallel. What does
1:02:11this mean? That there isn't just one
1:02:14DNA polymerase and one set of these
1:02:16nucleotides, but rather these
1:02:18components are there by the millions,
1:02:20and the template and primers are also
1:02:23there by the millions. So, let's
1:02:26imagine that the polymerization
1:02:31reaction begins, and then the primers
1:02:33—which we are not representing here
1:02:36—bind to the known region that was
1:02:39attached to one end of the molecule,
1:02:42and polymerization begins. Since most
1:02:46of the natural components are in excess
1:02:49, it is most likely that in the
1:02:50majority of the millions of molecules
1:02:52we have as a template, polymerization
1:02:54will occur completely. But in some of
1:02:58the molecules inside that tube, by
1:03:01chance, the DNA polymerase might place
1:03:04a dideoxy right at the first nucleotide
1:03:07to be polymerized. And for that
1:03:11molecule which was polymerized with
1:03:13that dideoxy, the polymerization of
1:03:15that molecule will end there. It will
1:03:17not be able to incorporate any more
1:03:18nucleotides. The same will happen with
1:03:22the second position. Most of the
1:03:25molecules continue their polymerization
1:03:27, but there will be a small subset that
1:03:30will, by chance, incorporate a dideoxy
1:03:33nucleotide at the second position of
1:03:35polymerization that is complementary to
1:03:38the template, and it will be
1:03:40polymerized, ending the polymerization.
1:03:45This will then occur consecutively at
1:03:47each of the polymerization positions,
1:03:50and when this reaction ends, we will
1:03:52have a complex mixture in that tube of
1:03:54fully polymerized molecules; that is,
1:03:57those that did not incorporate any
1:03:59dideoxy, but there will surely be a
1:04:01subset of molecules that have ended at
1:04:04every possible position within the
1:04:06polymerization. And this happens
1:04:10because there are millions and millions
1:04:12of molecules that participated in the
1:04:14polymerization. And so, statistically,
1:04:17it is most likely that at every
1:04:20position there is at least a subset of
1:04:23molecules represented. What does this
1:04:26mean? That all these molecules in which
1:04:30polymerization was interrupted at
1:04:32different parts will have different
1:04:34lengths. Hm. They will vary in length
1:04:38one by one. And we know that a
1:04:41technique to separate DNA molecules
1:04:44according to their size is
1:04:45electrophoresis. Hm. Electrophoresis in
1:04:49a porous matrix, for example, in an
1:04:51agarose gel or in a capillary that has
1:04:54a porous matrix. And by separating
1:04:59these fragments according to their size
1:05:02, each of the fragments, we will be
1:05:04able to identify which nucleotide was
1:05:06at the end, because each one of them
1:05:09emitted a fluorescent light depending
1:05:11on the fluorophore that was used,
1:05:13marking that so that, by analyzing them
1:05:18according to their size, the, uh, the
1:05:22light emitted by each of these
1:05:24fluorophores will be able to
1:05:27reconstruct the sequence of nucleotides
1:05:30that made up that fragment. This
1:05:34procedure is usually performed using
1:05:37not a common gel electrophoresis, but a
1:05:41capillary electrophoresis, just as we
1:05:44analyzed in the first part of the class
1:05:47. And the results are observed as these
1:05:52electropherograms, where we see a
1:05:56series of fluorescence peaks, and we
1:05:59now know that each position implies a
1:06:03fragment that differs by one nucleotide
1:06:06in length from the other. And the type
1:06:11of fluorescent light observed at each
1:06:14of these positions can be digitally
1:06:17decoded with the nature of one of the
1:06:21nucleotides, since the different
1:06:23fluorophores report each of the four
1:06:26DNA nucleotides. And in this way, we
1:06:30can digitally obtain, after this
1:06:33analysis of capillary electrophoresis,
1:06:36the nucleotide sequence, uh, of the
1:06:40fragment that was analyzed by Sanger
1:06:43sequencing. Let's observe this
1:06:47particularity. Let's think that we have
1:06:50obtained three different results
1:06:52through Sanger sequencing of a
1:06:54particular gene from three individuals.
1:06:59These three individuals have high
1:07:02similarities in this region, uh, of the
1:07:05particular gene that was analyzed, but
1:07:08the individual represented in the top
1:07:11electropherogram at this position has
1:07:14an adenine nucleotide. Hm. On the other
1:07:19hand, the individual represented in the
1:07:21bottom electropherogram, at this same
1:07:24position, has a guanine nucleotide.
1:07:26That is, this technique allows us, on
1:07:29one hand, to distinguish allelic
1:07:31variants that are present in different
1:07:33individuals, but I would like to point
1:07:36out the individual represented by the
1:07:38middle electropherogram, where two
1:07:40peaks appear simultaneously at this
1:07:43same position. One fluorophore
1:07:45reporting that there is adenine at that
1:07:47position and another fluorophore
1:07:49reporting that there is guanine at that
1:07:52same position. To correctly interpret
1:07:56this electropherogram, we have to
1:07:58remember that our genetic material is
1:08:01diploid. Therefore, it could happen
1:08:05that if the maternal and paternal
1:08:07allelic variants differ in their
1:08:10sequence, two different peaks will
1:08:12appear at the same position. In other
1:08:17words, this double peak being
1:08:19represented in the middle
1:08:21electropherogram is indicating an
1:08:24individual who has this allelic variant
1:08:27in heterozygosity. This Sanger
1:08:33sequencing method was widely used until
1:08:36the year 2000, but one of its
1:08:38particularities that can be thought of
1:08:40as a limitation is that for each Sanger
1:08:43experiment, only one type of molecule
1:08:45is sequenced. Right? For example, a
1:08:50particular exon of a gene, or an entire
1:08:52gene if it is small, can sometimes be
1:08:55sequenced. Hm. And while it has high
1:09:00sequencing fidelity, its costs are
1:09:03relatively high. Hm. This means it is
1:09:07only used if there is a clinical reason
1:09:11to suspect a relevant variant in a
1:09:14specific gene. Hm. Because only by
1:09:19being guided by clinical findings or
1:09:21this hypothesis will we go on to
1:09:23sequence that specific gene or exon. Hm
1:09:27. In other words, we would not apply
1:09:30the Sanger sequencing technique if we
1:09:32have a much more general hypothesis
1:09:34that there could be a pathogenic
1:09:36variant in the genome, but we do not
1:09:39know which gene is involved. For this
1:09:43other type of question, high-throughput
1:09:47sequencing methods might be more
1:09:49suitable than Sanger-based variants.
1:09:54What is the fundamental difference?
1:09:57Unlike Sanger, which sequenced a single
1:10:01type of molecule at a time, these
1:10:03methods sequence millions of molecules
1:10:07in parallel and could sequence, for
1:10:10example, an individual's entire genome
1:10:13at a relatively affordable cost and in
1:10:16much shorter times. Although we are not
1:10:23going to analyze the biochemistry of
1:10:26the high-throughput sequencing
1:10:29procedure, which is the goal of this
1:10:32course, it involves: extracting DNA
1:10:35from an individual, fragmenting these
1:10:38DNA molecules, sequencing the fragments
1:10:41in parallel, and then performing a
1:10:45bioinformatic analysis where the
1:10:48sequenced fragments are aligned with
1:10:51the complete reference genome available
1:10:55in public databases. This allows us to
1:10:58infer what differences were found in
1:11:01the individual from whom the DNA was
1:11:03extracted and subjected to this
1:11:05procedure, compared to the reference
1:11:08human genome. This methodology is
1:11:11increasingly being used in the context
1:11:14of medical genetics, but it has the
1:11:16difficulty that many variants are found
1:11:19with respect to the reference human
1:11:22genome. However, it is not always
1:11:24simple to determine if those variations
1:11:27found have a causal relationship with
1:11:30the pathology or if they are simply
1:11:33non-pathogenic variants that reflect
1:11:35the population's genetic diversity. Hm.
1:11:40I would finally like to mention
1:11:42regarding this technique that not only
1:11:44does it allow for whole-genome
1:11:46sequencing, but in some contexts,
1:11:48subsets of the genome can be sequenced.
1:11:51For example, we can focus only on the
1:11:54exonic regions of the genome, which are
1:11:57a much smaller percentage and allow us
1:12:00to focus on variations occurring in
1:12:02those coding regions of the genome. To
1:12:09finish this class, I would like to put
1:12:12into context what some situations might
1:12:16be where all the techniques we have
1:12:19analyzed today could be relevant. For
1:12:23example, let's consider we are
1:12:25performing a molecular diagnosis of
1:12:28monogenic entities. A first question is
1:12:31whether the clinical presentation, the
1:12:35signs and symptoms of the individual
1:12:38who consulted us, lead us to think
1:12:41there is a particular gene responsible
1:12:45for the signs and symptoms the
1:12:47individual presents. And based on prior
1:12:52history, because syndromes or clinical
1:12:55characteristics are already described
1:12:58that strongly suggest a pathology, we
1:13:01will be in certain conditions to make a
1:13:04molecular diagnosis that are different
1:13:07than if we do not have, for example, a
1:13:10strong hypothesis. If we don't have one
1:13:13, we probably have to use a
1:13:15hypothesis-free technique, right? An
1:13:18unbiased technique such as
1:13:20high-throughput sequencing. Hm. And
1:13:23there, we will eventually find, or not,
1:13:25variants in that individual that
1:13:28perhaps differ from the human reference
1:13:31genome. Hm. And we could develop a
1:13:34hypothesis regarding whether that
1:13:37variant is causal in producing the
1:13:40pathology. For example, we could
1:13:43corroborate that hypothesis if this
1:13:45same variant has been found in other
1:13:47individuals who have the same signs and
1:13:50symptoms, or if, in the context of
1:13:52basic research, some negative
1:13:54functional consequence of the
1:13:55appearance of that variant on protein
1:13:58function has been demonstrated; for
1:14:00example, if that variant is in a coding
1:14:02region. But let's imagine the other
1:14:07scenario where the signs and symptoms
1:14:10allow us to develop a hypothesis
1:14:12regarding which gene is involved in
1:14:15this disease. The next question is
1:14:20whether there are frequent mutations
1:14:22already described for that gene. And in
1:14:26the case where there are no frequently
1:14:28described mutations, one could perform
1:14:30Sanger sequencing of that gene and,
1:14:32again, see in that particular gene
1:14:34suggested by my clinical hypothesis if
1:14:36there are variants relative to the
1:14:38human reference genome or not, and if
1:14:40those variants have been reported as
1:14:43pathogenic or not. But in the case of
1:14:50having frequent mutations, or if it has
1:14:52already been found through other
1:14:54methodologies what mutations are
1:14:56present in a particular family, one
1:14:58could use more biased techniques. In
1:15:01other words, if I am looking for a
1:15:04particular mutation or allelic variant,
1:15:07I could use PCR-based techniques
1:15:09depending on the nature of that
1:15:11mutation. For example, we have seen
1:15:14that if the mutation we are looking for
1:15:17is an insertion or a deletion, we could
1:15:19perform conventional PCR. Given the
1:15:23difference in the size of the
1:15:24amplification products, the amplicons
1:15:26could report those allelic differences
1:15:28to us. We saw that in the case of
1:15:32substitution mutations, the appropriate
1:15:35variants of the PCR technique are, for
1:15:37example, allele-specific PCR or RFLP
1:15:40PCR. And in the particular case where
1:15:44our clinical context suggests a triplet
1:15:47expansion disease, we would use TPPCR,
1:15:50or Triple Prime PCR, since the other
1:15:53technique, uh, not based on PCR, the
1:15:56Southern blot technique, is one that,
1:15:59uh, while still used in some contexts,
1:16:02is increasingly falling into disuse due
1:16:06to its implementation difficulties. We
1:16:10also saw that uh or a variant of the
1:16:15PCR technique, specifically multiplex
1:16:17PCR, is used in the molecular context
1:16:20of forensic genetics. Here, there is no
1:16:24pathological entity involved; rather,
1:16:27the goal is to determine the profile of
1:16:30short tandem repeats, particularly
1:16:32microsatellites, in the context of
1:16:35parentage testing, for example, or
1:16:38other techniques associated with
1:16:40forensic genetics. With that, we finish
1:16:45today's class and we will see each
1:16:48other in a future session.

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