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Module 1: Ferramentas de DNA e Biotecnologia

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Nucleic Acid and Central Dogma

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A welcome to MOOC- NPTEL course on bioengineering an interface with biology and medicine, inlast 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 bioengineeringhas started making huge impact in many applications, then we kind of; we talked about basics especially,the live properties, the cell; difference cell organelles and their function.I also try to provide you a clinicians perspective by interaction with Dr. to give that you knowin which way clinicians also looking at various engineering solution that is logical solutionsfor various medical problems.So, this week we are going to talk mainly about the DNA and DNA tools and how biotechnologyhas started making impact by knowing the DNA technologies, so we are going to talk aboutyou know the various technologies involved in doing the molecular biology research especially,polymerase chain reaction, different gene cloning processes, those of all, we are goingto cover in this week.But I thought it will be important before we start going into detail of those technologiesto first introduce you again with the nucleic acids and central dogma that is the themefor today’s lecture although, I realised that you know you might have already studiedthis in your earlier courses, in other classes but just kind of refreshing you about someof the concept for the nucleic acids, gene and operon.And then, I will talk to you about central dogma and in which where it is important inthe 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 DNAtechnologies 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 componentsof DNA?So, the main function of the nucleic acid is to store and transmit the inter geneticinformation 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, deoxyribonucleicacid; DNA and ribonucleic acid or RNA.So, the major constituents of the chromosomes which are located in the nucleus of a cellis the DNA, which constitutes the genetic material.Whereas, the ribonucleic acid or RNA is the functional molecule or the working copy ofDNA which then participate in the process of protein synthesis, so from DNA to RNA theprocess of transcription happens and then from RNA to protein, process of translationhappens.Let me now, show you again and more so refresh you about the DNA structure and the componentand the Niels Bohr's 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 whichis cytosine, thymine, adenine and guanine; ATGC, you know cytosine and thymine, theseare 6 membered ring with 2 nitrogen which is having the pyrimidine ring structure.Or the adenine and guanine, they shed the structure with a purine ring and which ispyrimidine plus addition often, Imidazole ring.Now, Uracil is a; is unique because that is you know found in replacing thymine in caseof 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 ribosesugar and another is a deoxyribose sugar, both of them have the pentose sugar backboneand now, on the second carbon you can see there is hydroxyl OH group there in the ribosesugar, whereas in the deoxyribose, it is hydrogen, so that is the different between the riboseand deoxyribose and then third important component is a phosphate chain which is joined withthe; on the you know C5 carbon with hydroxyl of the sugar whether in case of ribose ordeoxyribose.And then because of this phosphate group, these nucleotides they are negatively chargedand that property is heavily used in the DNA electrophoresis.So, now let us kind of again look into these individual components, we talked about thebases, we talked about the sugar and we talked about the phosphate, right, these 3 componentstogether 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 nucleosideor 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 phosphodiesterbonds that is known as nucleic acids, so a nucleotide is a subunit of nucleic acid whichconsist of the nitrogen containing bases, which is having a 5 carbon sugar and the phosphategroup, 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 knowstraight 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 thecredit for the deducing the structure of the DNA and they are you know, how these are the2 strands are arranged in a helical form, how that are intertwined and the sugar phosphatebackbone lies on the outside whereas, the bases are inside and then the base pairs arespecifically 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 scientistyou know started working in that area and try to find out in which way the DNA structureis made.So, the Linus Pauling; he made a hypothesis that there are 3 chains, which are twistedaround each other and they form you know, some sort of rope like a strands and thatcould be you know, how the DNA structure is made, so then Wilkinson and Franklin, thesescientist; they provided x-ray crystallography data and then they found that you know, nucleotidesare 3.4 angstrom apart in the chain and the structure repeats at found that the 34 angstrominterval.So, there that gave much more clarity for the you know, the structure of DNA and thena scientist Chargaff, he provided the some basic rule that you know the if componentsare going to be equal to the T components of percentage of A = percentage of T and percentageof G = percentage of C base pairs.So, this summarises the kind of you know what we have discussed the structure of DNA inwhich 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 andCrick 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 replicationof DNA that is another interesting concept.So, the DNA double helix is actually you know, starts unwinding at the replication for, sonow 2 single strands are produced from this double helix DNA, which serves as templatesfor polymerisation of free nucleotides.Now, DNA polymer is; it starts polymerising these nucleotides by addition of some newnucleotides to the 3 prime end of the DNA chain and now, from the same DNA, now the2 copies of DNA is made.And as you can see that you know from the template S strand, now we have new strandbeing 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 youabout you know, the DNA replication and some of the classical experiments done in someother context that are on but just for the timing, I thought to you know, just give youthe 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 somuch fundamental to our life, to our hereditary information that you know how DNA structureis, is found and how you know it is compatible with all you know, any possible sequence ofbases 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 veryefficient means to store the genetic information, so knowing the DNA structure becomes verycrucial and the DNA sequence actually ultimately determines the sequence of the you know, ribonucleicacid and eventually, the proteins are formed from that so, these sequences of bases alongwith one strand they are you know, completely determines the sequence of other strand.And then, they are going to dictate the RNA formation and protein formation, so afteryou know revealing some basic concepts of DNA and DNA structure, let us now think abouta question about what is the gene, right.The concept of gene has actually evolved through the history of genetics starting from youknow the scientists like Mendel who was thinking about you know, there is some hereditary factorswhich are; he did not know about gene that these formations are passing from one to nextgeneration, probably there are some factors which are involved, which are having theseinformation is stored.And then, Morgan; kind of provided some further experimental evidences that you know thesehereditary units are actually located on the chromosomes, so many scientists have contributedin the journey and now, finally we know that you know what we consider a gene is actuallya discrete unit of inheritance or you can also defined that it is a region of a specificnucleotide sequences in a chromosome.Or you can say, it is the DNA sequence that codes for a specific polypeptide chain, soif 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, itcan 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 havingyou know, many processes which are involving to; to shuffle from the DNA to make the RNAbut kind of this try to illustrate you are that we have you know various exons regionsand we have a various introns, now in the; in the process of alternative splicing inwhich way now these introns are removed and the coded form the exons are coming togetherto give rise to the functional RNA molecule.So, there are multiple control elements, which are actually associated with the eukaryoticgenes and the segments of non-coding DNA which help to regulate the transcription by bindingto 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 anisomer of lactose sugar that you know, D represents the operon by inactivating the repressor andin this manner, and this enzyme for the lactose could be utilised and then it can be furtherinduced, so I am going to show you this in one of the animation and to explain you inmuch more detail.In prokaryotes, transcription by RNA polymerase can take place with the help of an activatorprotein.However, in the presence of a repressor molecule, the binding site for RNA polymerase is inaccessibledue to which transcription does not occur.In the ground state, the repressor does not remain bound because of which the gene isturned own.The lac operon consists of a group of genes that are responsible for transport and metabolismof lactose sugar in certain bacteria like E. coli, this operon is under negative regulationby the lack I; repressor protein.In absence of inducer, the tetrameric repressor binds to the operator region thereby, preventingtranscription by RNA polymerase.In presence of inducer, the inducer binds to the repressor protein, which then preventsit from binding to the operator and therefore, allows gene expression.An inducible system is off state in its ground state and must be turned on by an effectivemolecule, which is known as inducer.In positive regulation mechanism however, the inducer binds to the inactive activatorto produce the active activator molecule which in turn facilitates binding of RNA polymeraseto the promoter to turn on expression.In the negative regulation mechanism, the inducer binds to repressor and prevents itfrom binding to the operator region; this allows RNA polymerase to proceed with transcriptionby binding to the promoter.The ground state in case of repressible system is own, it has to be turned off by an effectormolecule which is known as a co-repressor.In positive regulation, the co- repressor binds to the activator molecule and preventsits binding to the promoter region thereby, turning of gene expression.In case of negative regulation mechanism, the co-repressor binds to the inactive repressormolecule and activates it thereby, preventing gene expression.The lac operon consists of a group of genes that are responsible for transport and metabolismof lactose sugar in certain bacteria like E. coli, this operon is under negative regulationby the lack I repressor protein, in absence of inducer, the tetrameric repressor bindsto the operator region thereby, preventing transcription by RNA polymerase.In presence of the inducer, the inducer binds to the repressor protein, which then preventsit 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 levelsprevent synthesis of the cyclic AMP which is essential for binding to the cataboliteactivator protein, this protein facilitates transcription of the lac operon.When glucose levels are low, cyclic AMP is produced, which binds to this gap which inturn 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 whichway it regulate the synthesis of inducible enzyme, okay.Now, let us move on to you know, thinking about from the cell, where we can find theDNA, right, so let us say the human body is made of billions and trillions of cells, wehave discussed in the last class as well.And then each of those cell is having these you know, the nucleus which contains the geneticmaterial, now each cell contains 2 copies of these chromosomes and now, these chromosomesif you expand further, you can see the long DNA molecules, even the genes and the functionregion of DNA, we can; we can see over there and then, now you can see the tiny pictureof you know how these DNA molecules you know, if you think about the cells, each cell havingthe 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 isso crucial which dictates all the hereditary information and it is you know, kind of packagingin this cell becomes very crucial as well and again, to refresh you from the previouslectures in the cell context if you think you know how the nucleic acid contents areso tightly packed inside the nucleus in a small area with you know very intricate youknow binding with histone proteins.So, these histone proteins and these DNA molecules they formed these nucleosomes and these nucleosomestogether are packed to form the chromatins, this how you know, these particular packaginghappens inside the nucleus and let thinking about eukaryotic genomes, how they are organisingthe 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 chargedproteins which could interact with the DNA molecule which is negatively charged and thatactually helps to compact the DNA and these nucleosomes could be seen like the beads ona 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 sexchromosome; X or Y chromosome.And a process known as karyotyping, where you want to look at the pattern of each ofthe chromosomes tells us about the you know, is the pattern of these chromosomes are normalor 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 thereis some sort of syndrome is present like down syndrome or some sort of you know the issueswith 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 butideally, it shows us the organisation for the whole genome, alright, so there have beenmany discoveries which have contributed immensely to the field of you know, overall DNA relateddiscovery 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 goingto talk in much more detail about you know many of these fundamentals and their applicationsin subsequent lectures.But just to kind of you know, refresh you and bring to the scale, starting from GregorMendel, the father of genetics, 1822 to 1844, lot of elegant experiment being done on thepea plant, which gives us the; the basic idea for you know the; how various laws of hereditaryare governed and then with the ideas for discrete factors which Mendel mention, the genes areactually 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 illustratedthe structure of DNA.And but you know the initial part from 1865 for Mendel is you know gets credit and Mendelis known as the father of genetics for his contribution.Then, as you go on the time a scale, a Sturtevant, he made the first linear map of the genesin 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 develpoed DNA sequencing methods in 1977.In 1983, the first human disease gene was mapped with the DNA markers especially inthe 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 knowingabout all the human genes in 1988 and then while those things were happening, we startedknowing 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 1997and that is another you know one of the scientific fiction and an story in which one could producea 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 accomplishmentat 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 natureand science and you know, those project actually, help us to, to really try to get a biggerpicture of what is happening inside you know, the entire human genome, what are all genespresent there and you know it is the first most ambitious project to really understandyou know beyond moving on to the single gene and looking at you know just the characteristicsgoverned 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 communityin which way we are able to now of work you know for the understanding the all the moleculesof life.For example, Omics molecules, so this brings to the second part which is thinking aboutcentral dogma.So, now we have studied about DNA, which is the genetic blueprint and just you know imaginethat in a cell, now you want to; first of all you want to know that you know where theDNA 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 proteinshas 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 poor area in IIT Bombay and we want to makea building campus here, so from the map now you know that you know where the DNA, thegenetic blueprint and in that area then, some contractors will come and then they will tryto you know make a map that where the building has to be made and then the proteins are thebuilding 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 have wide good technology in which wayDNA to RNA to proteins, everything is crucial but in which way the proteins are much moreyou know direct in function they are providing much more functional molecules while the geneticsblueprint come from the DNA.Now, let us see you know kind of briefly refresh the class of RNA’s, we have the messengerRNAs, we have transfer RNAs and ribosomal RNAs.Messenger RNAs; they provide a template for protein synthesis for or the transition processto happen but they are very less abundant only you know 5% population is there for themRNA, where the transfer RNA’s, they are the carriers of amino acids and activate toform ribosomes.Then the ribosomal RNA’s, they are the major component of ribosomes, they provide catalyticand the structural roles and there actually, the most abundant among the RNA population,so RNA’s, they are involved in the protein synthesis process, again which is say, youknow an interesting but the complex subject which needs much more you know full lecturebut just to brief you, the mRNA provides a long sequence of nucleotides, which servesas a template for the protein synthesis to happen.Now, the tRNA’s they are involved in the protein synthesis but that binds an aminoacid at one end and the base pairs with a mRNA codon on the other hand and then thatserves 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 andthe structural rules in the protein synthesis process.So, transcription and translation again you know, there is lot of fundamentals involvedin understanding these processes, this slide just kind of gives you the illustration thatin which way from DNA the RNAs are being formed in the process of transcription and then fromthere in which way whereas, amino acids are formed in the translation process happensto generate the proteins.So, this is you know, we will talk as you go along but I just want to convey you thatyou know these orderly and unidirectional flow of information happens in the cell asa part of central dogma and that actually that information is in the base sequence ofthe DNA, which flows from DNA to RNA to the protein and this is what we say central dogmawhich involves 2 important steps of transcription and translation.And of course, you know you are also aware that there could be reverse transcriptionas well but the information could also flow from RNA to DNA and that is also you knowvery crucial for many biological phenomenon to happen.An important thing you know, why from the same gene, we still see different type ofRNA forms.And by multiple protein forms you know becomes very crucial to understand and that is actuallybeing dictated by 2 important phenomenon; one is alternative splicing, so in alternativesplicing that is a process in which the exons or the coding sequence of pre-mRNA, they areproduced by the transcription of a gene and then there are combined in different waysduring RNA splicing.So, what happens then, the resulting mature mRNA, it gives rise to different protein productsas the part of translation.And then, they are the isoforms of one another, so now you have the single gene but actuallythat can give rise to multiple you know RNA forms in different protein products, so asyou can see in the picture from the one of the pre-mRNA, we have mature mRNA-A and maturemRNA-B, they are being formed and they give rise to the red colour protein, protein Aor the green colour protein, protein B.Then, after the proteins have been synthesised then further modification may happen at theprotein level and that is known as the post-translational modifications, so many proteins undergo post-translationmodification at some of their amino acid residues and some you know, molecules could be addedfor the sugar moieties as the part of glucosylation or phosphate moiety as the part of the phosphorylationor it can be hydroxylation, methylation, alkylation, acetylation.And many kinds of modifications may happen which makes the protein very different functionallyand that is where you know studying the RNA molecules or studying protein molecules providemuch more function and formation because many of these modification are actually quite relevantthinking about the actual physiological question, right, these information what I just conveyedyou 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 allthe genes of a given organism of a given system, let us say for human, we have no idea forthe entire genome and that we say the human genome project or human genome sequences areavailable now and similarly, do we have idea for the entire human transcriptome or humanproteome and that can help us to really understand the system much better as compared to thinkingabout just 1 or 2 protein at that time.So, then the Omics understanding that so this whole field is known as Omics field, whichaims to look at all the molecules present you know in a given system and then this informationcould be very valuable and useful for the patient's treatment and patient therapy, thinkabout the personalised medicine which is an area upcoming right now, where intention isto look at these biomolecules you know from a given individual and use that entire informationfor their treatment.So, whether you think about you know having the diagnostic test or think about you knowthe integrating these personalised interventions or optimising them over the period for thetreatment modalities, all of them requires good understanding of you know these basicbiomolecules 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, theDNA and RNA, we also try to cover briefly about histones and in which way the DNA arebeing packed, then I try to convey you there that in which were central dogma, flows informationfrom DNA to RNA in the process of transcription and then protein in the form of translationand then in which way the new emerging field of Omics technology is able to you know isactually aiming to understand the complex signalling pathways, which could be involvedin the biological system.And that may lead us to do better cure at the personalised medicine level, so theseare all some of the basic fundamental of course, you know to know more in detail, you haveto either take some more advanced classes or you have to you know take more specialisedbiological classes but our intention was to give you some overview to refresh about thesebiomolecules.And now, we are set to talk to you about various advances which are happening in the DNA toolsand biotechnology area and that will be the main focus for the next of the lectures forthis week.Thank you very much.