Week 2-Lecture 6

Week 2-Lecture 6


A welcome to MOOC- NPTEL course on bioengineering
an interface with biology and medicine, in last week, we started discussing about why
biology is so important for engineering discipline, I try to give you various examples in which
we; we can see that you know the bioengineering has started making huge impact in many applications,
then we kind of; we talked about basics especially, the live properties, the cell; different cell
organelles and their function. I also try to provide you a clinician’s
perspective by interaction with Dr. Aliasgar Moiyadi to give you understanding that in
which way clinicians also looking at various engineering solution, technological logical
solutions for various medical problems. So, this week we are going to talk mainly
about the DNA and DNA tools and how biotechnology has started making impact by knowing the DNA
technologies, so we are going to talk about you know the various technologies involved
in doing the molecular biology research especially, polymerase chain reaction, different gene
cloning processes, those of all, we are going to cover in this week. But I thought it will be important before
we start going into detail of those technologies to first introduce you again with the nucleic
acids and central dogma that is the theme for today’s lecture although, I realised
that you know you might have already studied this in your earlier courses, in other classes
but just kind of refreshing you about some of the concepts for the nucleic acids, gene
and operon. And then, I will talk to you about central
dogma and in which where it is important in the Omics era and then these concepts, how
we can you know, try to utilise these understanding, then in the next; next set of lectures in
this whole week is going to utilise a DNA technologies and going to illustrate you in
which we way can do research in these areas. Let us first start with the basic concept
of nucleic acid, which are the basic components of DNA? So, the main function of the nucleic acid
is to store and transmit the entire genetic information and there are you know, 2 specific
classes of sugars based on which the entire, the classification happens for nucleic acid,
which is DNA or RNA, right, deoxyribonucleic acid; DNA and ribonucleic acid or RNA. So, the major constituents of the chromosomes
which are located in the nucleus of a cell is the DNA, which constitutes the genetic
material. Whereas, the ribonucleic acid or RNA is the
functional molecule or the working copy of DNA which then participate in the process
of protein synthesis, so from DNA to RNA the process of transcription happens and then
from RNA to protein, process of translation happens. Let me now, show you again and more so refresh
you about the DNA structure and the component and those involved in making DNA structure,
so for example there are 4 bases; the monomers, which constitutes DNA and these are the structure
shown on the screen which is cytosine, thymine, adenine and guanine; ATGC, you know cytosine
and thymine, these are 6 membered ring with 2 nitrogen which is having the pyrimidine
ring structure. Or the adenine and guanine, they share the
structure with a purine ring and which is pyrimidine plus addition often, Imidazole
ring. Now, Uracil is a; is unique because that is
you know found in replacing thymine in case of RNA, so you can look at the structure of
Uracil here. Now, let us talk about briefly these sugars;
there are 2 sugars involved, one is the ribose sugar and another is a deoxyribose sugar,
both of them have the pentose sugar backbone and now, on the second carbon you can see
there is hydroxyl OH group there in the ribose sugar, whereas in the deoxyribose, it is hydrogen,
so that is the difference between the ribose and deoxyribose and then third important component
is a phosphate chain which is joined with the; on the you know C5 carbon with hydroxyl
of the sugar whether in case of ribose or deoxyribose. And then because of this phosphate group,
these nucleotides they are negatively charged and that property is heavily used in the DNA
electrophoresis. So, now let us kind of again look into these
individual components, we talked about the bases, we talked about the sugar and we talked
about the phosphate, right, these 3 components together form the nucleic acids and let us
look at some terminology for example, nucleoside, when you are combining a base and the sugar
form that together gives rise to nucleoside or when we add a base, sugar and phosphate
chain that is nucleotide. And then, when you are combining many nucleotides
which are actually join with the phosphodiester bonds that is known as nucleic acids, so a
nucleotide is a subunit of nucleic acid which consist of the nitrogen containing bases,
which is having the 5 carbon sugar and the phosphate group, so again the structure is
shown you here, which is for a nucleotide. So, now in this way now, you can easily decode
the entire DNA structure which is you know straight forward know, we have good understanding
that you know in which the nitrogen bases, the sugar phosphate backbone is constituting
the DNA structure. But of course, it was not known earlier and
the scientist Watson and Crick, they get the credit for the deducing the structure of the
DNA and they are you know, how these are the 2 strands are arranged in a helical form,
how that are intertwined and the sugar phosphate backbone lies on the outside whereas, the
bases are inside and then the base pairs are specifically forming the bonds, which is hydrogen
bonds between A and T and G and C bases, right. So, there was always that you know quest to
elucidate the structure of DNA and many scientists you know started working in that area and
try to find out in which way the DNA structure is made. So, the Linus Pauling; he made a hypothesis
that there are 3 chains, which are twisted around each other and they form you know,
some sort of rope like a strands and that could be you know, how the DNA structure is
made, so then Wilkinson and Franklin, these scientist; they provided x-ray crystallography
data and then they found that you know, nucleotides are 3.4 angstrom apart in the chain and the
structure repeats at found that the 34 angstrom interval. So, there that gave much more clarity for
the you know, the structure of DNA and then a scientist Chargaff, he provided the some
basic rule that you know the if components are going to be equal to the T components
of percentage of A=percentage of T and percentage of G=percentage of C base pairs. So, this summarises the kind of you know what
we have discussed the structure of DNA in which way, adenine and thymine are they form
the 2 hydrogen bonds and guanine and cytosine, they form 3 hydrogen bonds and the structure
of DNA decoded by a scientist, Watson and Crick for which they were awarded the Nobel
Prize in 1962. Now, then you know, how DNA makes its multiple
copies, so the DNA duplication or replication of DNA that is another interesting concept. So, the DNA double helix is actually you know,
starts unwinding at the replication for, so now 2 single strands are produced from this
double helix DNA, which serves as templates for polymerisation of free nucleotides. Now, DNA polymer is; it starts polymerising
these nucleotides by addition of some new nucleotides to the 3 prime end of the DNA
chain and now, from the same DNA, now the 2 copies of DNA is made. And as you can see that you know from the
template S strand, now we have new strand being synthesised and now, we got 2 DNA molecules
in the process of replication. So, the different theories which are involved
in the DNA replication, I will talk to you about you know, the DNA replication and some
of the classical experiments done in some other context later on but just for the timing,
I thought to you know, just give you the feel of that you know, how DNA copies are being
made. And why it is so important for us to understand
DNA structure because you know, the DNA so much fundamental to our life, to our hereditary
information that you know how DNA structure is, is found and how you know it is compatible
with all you know, any possible sequence of bases that is very important, I think for
us to appreciate. And understand that the sequences of bases
along any DNA strand, they acts as a very efficient means to store the genetic information,
so knowing the DNA structure becomes very crucial and the DNA sequence actually ultimately
determines the sequence of the you know, ribonucleic acid and eventually, the proteins are formed
from that so, these sequences of bases along with one strand they are you know, completely
determines the sequence of other strand. And then, they are going to dictate the RNA
formation and the protein formation, so after you know revealing some basic concepts of
DNA and DNA structure, let us now think about a question about what is the gene, right. The concept of gene has actually evolved through
the history of genetics starting from you know the scientists like Mendel who was thinking
about you know, there is some hereditary factors which are; he did not know about gene that
these formations are passing from one to next generation, probably there are some factors
which are involved, which are having these information is stored. And then, Morgan; kind of provided some further
experimental evidences that you know these hereditary units are actually located on the
chromosomes, so many scientists have contributed in the journey and now, finally we know that
you know what we consider a gene is actually a discrete unit of inheritance or you can
also defined that it is a region of a specific nucleotide sequences in a chromosome. Or you can say, it is the DNA sequence that
codes for a specific polypeptide chain, so if you just want to you know, get a broad
overview of gene, I think we can summarise, it is a region of DNA that can be expressed
to produce a final functional product, it can be a polypeptide or it can be an RNA molecule
so, this is how you can think about a gene, one of the discrete unit of inheritance, which
is providing these kind of function and formation. Now, the organisation of a typical eukaryotic
gene is you know is really complex and having you know, many processes which are involving
to; to shuffle from the DNA to make the RNA but kind of this try to illustrate you are
that we have you know various exons regions and we have a various introns, now in the;
in the process of alternative splicing in which way now these introns are removed and
the coded form the exons are coming together to give rise to the functional RNA molecule. So, there are multiple control elements, which
are actually associated with the eukaryotic genes and these are the segments of non-coding
DNA which help to regulate the transcription by binding to the certain proteins. The concept of Lac operon becomes very crucial. In this model, you can see that you know in
the; if you have Allolactose, which is an isomer of lactose sugar that you know, derepresses
the operon by inactivating the repressor and in this manner, and this enzyme for the lactose
could be utilised and then it can be further induced. So I am going to show you this in one of the
animation and to explain you in much more detail. In prokaryotes, transcription by RNA polymerase
can take place with the help of an activator protein. However, in the presence of a repressor molecule,
the binding site for RNA polymerase is inaccessible due to which transcription does not occur. In the ground state, the repressor does not
remain bound because of which the gene is turned on. The lac operon consists of a group of genes
that are responsible for transport and metabolism of lactose sugar in certain bacteria like
E. coli, this operon is under negative regulation by the lack I; repressor protein. In absence of inducer, the tetrameric repressor
binds to the operator region thereby, preventing transcription by RNA polymerase. In presence of inducer, the inducer binds
to the repressor protein, which then prevents it from binding to the operator and therefore,
allows gene expression. An inducible system is off in its ground state
and must be turned on by an effective molecule, which is known as inducer. In positive regulation mechanism however,
the inducer binds to the inactive activator to produce the active activator molecule which
in turn facilitates binding of RNA polymerase to the promoter to turn on expression. In the negative regulation mechanism, the
inducer binds to repressor and prevents it from binding to the operator region; this
allows RNA polymerase to proceed with transcription by binding to the promoter. The ground state in case of repressible system
is on, it has to be turned off by an effector molecule which is known as a co-repressor. In positive regulation, the co- repressor
binds to the activator molecule and prevents its binding to the promoter region thereby,
turning off gene expression. In case of negative regulation mechanism,
the co-repressor binds to the inactive repressor molecule and activates it thereby, preventing
gene expression. The lac operon consists of a group of genes
that are responsible for transport and metabolism of lactose sugar in certain bacteria like
E. coli, this operon is under negative regulation by the lack I repressor protein, in absence
of inducer, the tetrameric repressor binds to the operator region thereby, preventing
transcription by RNA polymerase. In presence of the inducer, the inducer binds
to the repressor protein, which then prevents it from binding to the operator and therefore,
allows gene expression. Lac operon also undergoes positive regulation
by means of the cyclic AMP Cap system. Glucose is a preferred energy source for bacteria
and if both glucose and lactose are present, beta galactosidase enzyme, which metabolises
lactose is not synthesise, high glucose levels prevent synthesis of the cyclic AMP which
is essential for binding to the catabolite activator protein, this protein facilitates
transcription of the lac operon. When glucose levels are low, cyclic AMP is
produced, which binds to this gap which in turn binds to a distal part of the promoter
region and facilitates transcription. But this kind of you know, the slide illustrates
you the broad model of lac operon and in which way it regulates the synthesis of inducible
enzyme, okay. Now, let us move on to you know, thinking
about from the cell, where we can find the DNA, right, so let us say the human body is
made of billions and trillions of cells, we have discussed in the last class as well. And then each of those cell is having these
you know, the nucleus which contains the genetic material, now each cell contains 2 copies
of these chromosomes and now, these chromosomes if you expand further, you can see the long
DNA molecules, even the genes and the function region of DNA, we can; we can see over there
and then, now you can see the tiny picture of you know how these DNA molecules you know,
if you think about the cells, each cell having the nucleus, having the chromosomes, having
the gene and their; the DNA part. So, this is you know, really tiny bit of the
molecule present in the cell but that it is so crucial which dictates all the hereditary
information and it is you know, kind of packaging in this cell becomes very crucial as well
and again, to refresh you from the previous lectures in the cell context if you think
you know how the nucleic acid contents are so tightly packed inside the nucleus in a
small area with you know very intricate you know binding with histone proteins. So, these histone proteins and these DNA molecules
they formed these nucleosomes and these nucleosomes together are packed to form the chromatins,
this how you know, these particular packaging happens inside the nucleus and let thinking
about eukaryotic genomes, how they are organising the chromosomes, I think you know, knowing
about the histone proteins becomes very crucial. So, these histone proteins as I mention H2A,
H2B, H3 and H4, they are positively charged proteins which could interact with the DNA
molecule which is negatively charged and that actually helps to compact the DNA and these
nucleosomes could be seen like the beads on a string, so this is how you can; you can
think about from DNA to chromosomes. A chromosomes consist of a DNA molecule which
is packed together with the proteins and now, these chromosomes could be seen which are
having these you know, bead kind of a structures, right, alright, so human having 23 pair of
chromosomes, 22 autosomes + one pair of sex chromosome; X or Y chromosome. And a process known as karyotyping, where
you want to look at the pattern of each of the chromosomes tells us about the you know,
is the pattern of these chromosomes are normal or is there any abnormality can be seen and
for many disorders like especially you know, chromosomal aberrations can be found and that
actually, helps us to; to deduce is there is some sort of syndrome is present like down
syndrome or some sort of you know the issues with any other chromosomes, abnormalities
are there. And even for sex determination, people look
at the X and the Y chromosomes and their patterns, so this image just shows you know the colour
painting of these various chromosomes but ideally, it shows us the organisation for
the whole genome, all right, so there have been many discoveries which have contributed
immensely to the field of you know, overall DNA related discovery which have contributed
entirely from genetics to genomics areas. I am going to show you couple of mile stones
discoveries although, you know we are going to talk in much more detail about you know
many of these fundamentals and their applications in subsequent lectures. But just to kind of you know, refresh you
and bring to the scale, starting from Gregor Mendel, the father of genetics, 1822 to 1884,
lot of elegant experiment being done on the pea plant, which gives us the; the basic idea
for you know the; how various laws of hereditary are governed and then with the ideas for discrete
factors which Mendel mention, the genes are actually going to transmit characteristics
from one generation to the other generation. Over the period, then we had you know the
discoveries by Watson and Crick, which illustrated the structure of DNA. And but you know the initial part from 1865
for Mendel is you know gets credit and Mendel is known as the father of genetics for his
contribution. Then, as you go on the time a scale, Sturtevant,
he made the first linear map of the genes in 1913. And then, came the Watson and Crick contribution
for double helical structure of DNA in 1953. Then scientist Nirenberg, Khorana and Holly,
they first mention the genetic code in 1966. Scientist Cohen and Boyer, they developed
recombinant DNA technology in 1972. And then, Sangar, Maxan and Gilbert, they
developed DNA sequencing methods in 1977. In 1983, the first human disease gene was
mapped with the DNA markers especially in the disease of Huntington disease are shown. And you know, one of the milestone technologies
polymer chain reaction was invented in 1985. And then, the human genome organisation started
you know, an ambitious project of knowing about all the human genes in 1988 and then
while those things were happening, we started knowing more about the cell, about cloning,
about you know development process and reprogramming. And that eventually, culminated into the cloning
of an animal, Dolly by Ian Wilmut in 1997 and that is another you know one of the scientific
fiction and an story in which one could produce a cell or organism with the same nuclear genome
as another cellular organism. And Dr. Ian Wilmut of Roslin institute, they
cloned this sheep Dolly which was major accomplishment at that time. Then human genome sequence projects are getting
competed in the years 2001 to 2003. And then, first draft of the human genome
map was presented in cover page of nature and science and you know, those project actually,
help us to, to really try to get a bigger picture of what is happening inside you know,
the entire human genome, what are all genes present there and you know it is the first
most ambitious project to really understand you know beyond moving on to the single gene
and looking at you know just the characteristics governed from a gene that what is happening
in the entire genome. And all the gene, how they are governing the
function, so that kind of you know was the; as the big accomplishment not only to understand
the gene but also in the scientific community in which way we are able to now of work you
know for the understanding the all the molecules of life. For example, Omics molecules, so this brings
to the second part which is thinking about central dogma. So, now we have studied about DNA, which is
the genetic blueprint and just you know imagine that in a cell, now you want to; first of
all, you want to know that you know where the DNA is and that DNA is going to make the
molecular photocopy which is RNA and that is you know, like the functional molecule
has to be initiated and from those RNA molecules, the proteins has to be made, so now use the
same analogy for let us say making a building. So, let us say you know we are in Mumbai in
Powai area in IIT Bombay and we want to make a building campus here, so from the map now
you know that you know where the DNA, the genetic blueprint and in that area then, some
contractors will come and then they will try to you know make a map that where the building
has to be made and then the proteins are the building material will come which is going
to be like you know, the mortars and bricks, which is going to create that building which
is you know like the engines of biology. So this something you know which helps you
to draw analogy in which way DNA to RNA to proteins, everything is crucial but in which
way the proteins are much more you know direct in function they are providing much more functional
molecules while the genetics blueprint come from the DNA. Now, let us see you know kind of briefly refresh
the class of RNA’s, we have the messenger RNAs, we have transfer RNAs and ribosomal
RNAs. Messenger RNAs; they provide a template for
protein synthesis for or the transition process to happen but they are very less abundant
only you know 5% population is there for the mRNA, where the transfer RNA’s, they are
the carriers of amino acids and activated form to ribosomes. Then the ribosomal RNA’s, they are the major
component of ribosomes, they provide catalytic and the structural roles and they are actually,
the most abundant among the RNA population, so RNA’s, they are involved in the protein
synthesis process, again which is say, you know an interesting but the complex subject
which needs much more you know full lecture but just to brief you, the mRNA provides a
long sequence of nucleotides, which serves as a template for the protein synthesis to
happen. Now, the tRNA’s they are involved in the
protein synthesis but that binds an amino acid at one end and the base pairs with a
mRNA codon on the other hand and then that serves as an adapter that translates mRNA
code into a sequence of amino acids. Then rRNA, it forms the central components
of ribosomes, it also plays catalytic and the structural rules in the protein synthesis
process. So, transcription and translation again you
know, there is lot of fundamentals involved in understanding these processes, this slide
just kind of gives you the illustration that in which way from DNA the RNAs are being formed
in the process of transcription and then from there in which way whereas, amino acids are
formed in the translation process happens to generate the proteins. So, this is you know, we will talk as you
go along but I just want to convey you that you know these orderly and unidirectional
flow of information happens in the cell as a part of central dogma and that actually
that information is in the base sequence of the DNA, which flows from DNA to RNA to the
protein and this is what we say central dogma which involves 2 important steps of transcription
and translation. And of course, you know you are also aware
that there could be reverse transcription as well but the information could also flow
from RNA to DNA and that is also you know very crucial for many biological phenomenon
to happen. An important thing you know, why from the
same gene, we still see different type of RNA forms. And by multiple protein forms you know becomes
very crucial to understand and that is actually being dictated by 2 important phenomenon;
one is alternative splicing, so in alternative splicing that is a process in which the exons
or the coding sequence of pre-mRNA, they are produced by the transcription of a gene and
then they are combined in different ways during RNA splicing. So, what happens then, the resulting mature
mRNA, it gives rise to different protein products as the part of translation. And then, they are the isoforms of one another,
so now you have the single gene but actually that can give rise to multiple you know RNA
forms in different protein products, so as you can see in the picture from the one of
the pre-mRNA, we have mature mRNA-A and mature mRNA-B, they are being formed and they give
rise to the red colour protein, protein A or the green colour protein, protein B. Then, after the proteins have been synthesised
then further modification may happen at the protein level and that is known as the post-translational
modifications, so many proteins undergo post-translation modification at some of their amino acid residues
and some you know, molecules could be added for the sugar moieties as the part of glucosylation
or phosphate moiety as the part of the phosphorylation or it can be hydroxylation, methylation, alkylation,
acetylation. And many kinds of modifications may happen
which makes the protein very different functionally and that is where you know studying the RNA
molecules or studying protein molecules provide much more function and formation because many
of these modification are actually quite relevant thinking about the actual physiological question,
right, so these information what I just conveyed you are looking at the central dogma, all
the genes, then the transcripts and the proteins. Now, scientists are trying to study in much
more totality for example, can we study all the genes of a given organism of a given system,
let us say for human, we have no idea for the entire genome and that we say the human
genome project or human genome sequences are available now and similarly, do we have idea
for the entire human transcriptome or human proteome and that can help us to really understand
the system much better as compared to thinking about just 1 or 2 protein at that time. So, then the Omics understanding that so this
whole field is known as Omics field, which aims to look at all the molecules present
you know in a given system and then this information could be very valuable and useful for the
patient’s treatment and patient therapy, think about the personalised medicine which is an
area upcoming right now, where intention is to look at these biomolecules you know from
a given individual and use that entire information for their treatment. So, whether you think about you know having
the diagnostic test or think about you know the integrating these personalised interventions
or optimising them over the period for the treatment modalities, all of them requires
good understanding of you know these basic biomolecules and if possible, the technology
to understand them at the OMICS level. So, in summary today we just talked about
the basics of nucleic acid especially, the DNA and RNA, we also try to cover briefly
about histones and in which way the DNA are being packed, then I try to convey you there
that in which were central dogma, flows information from DNA to RNA in the process of transcription
and then protein in the form of translation and then in which way the new emerging field
of Omics technology is able to you know is actually aiming to understand the complex
signalling pathways, which could be involved in the biological system. And that may lead us to do better cure at
the personalised medicine level, so these are all some of the basic fundamental of course,
you know to know more in detail, you have to either take some more advanced classes
or you have to you know take more specialised biological classes but our intention was to
give you some overview to refresh about these biomolecules. And now, we are set to talk to you about various
advances which are happening in the DNA tools and biotechnology area and that will be the
main focus for the next of the lectures for this week. Thank you very much.

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