#15 nucleic acids and their detection.pdf - McGill BIOL200 Fall 2019 Lecture 16 Nucleic acids and their detection Chapter 6 pages 246-247 can sense

#15 nucleic acids and their detection.pdf - McGill BIOL200...

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Unformatted text preview: McGill BIOL200 - Fall 2019 Lecture 16: Nucleic acids and their detection Chapter 6: pages 246-247 can sense things in our environment and its those sense thats somehow tricked down and affect our germ cells so that it somehow affects the upcoming generations. refer to this as transgeneration epigenetic inheritance. going to discuss intricacies involved with molecular biology, gene regulation and transcription. © R. Roy, 2019 McGill BIOL200 - Fall 2019 Molecular biological techniques advance our analytical capabilities introduce to methods we can use in order to better characterize and analyze nucleic acids (RNA and DNA). by using these methods, we can be quantitative and qualitative. both of these aspects of molecular bio are important QUALITATIVE ANALYSIS-the nature of the molecule(s) in question. -size? giant molecule ? little? does it change? sequence and changes in sequences can have fundamental conseuences -nucleotide composition? for the biology of all organisms. -conformation/configuration? because sequences is so important -structure? depending on what perspective we might have, structure becomes very important QUANTITATIVE ANALYSIS-determine the levels of specific gene products. ie…tumour markers (p53, BRCA1/2) sometimes we need to know absolute levels of certain gene products, these gene products might include things like oncogenic products or tumor suppresors or various other factors that might help physicians make a diagnosis or prognosis. by understanding the levels of certain nucleic acids, we can get a better idea of how that might contribute to some sort of homeostatic state. © R. Roy, 2019 McGill BIOL200 - Fall 2019 Molecular Probes can be used to find a needle in a haystack… A complex mixture of macromolecules Binding to a membrane Probe specific for target ( ) Nitrocellulose or Nylon Remove non-specific Target Detection © R. Roy, 2019 molecular bio in lab, very often sometimes you just have to believe because somtimes youre looking at bottom of test tubes, and people say DNA is there but you say there isnt. its there… molecular biol is like homeopathy, have to believe but really what makes us believe, or helps us to conclude is the fact that we have these extraordinary tools to actually tell us that in the bottom of that test tube, is in fact the molecule that you had hoped/ believed or were trying to achieve. the way we can do that with minute quantities of DNA and RNA is by making specific detectors that allow us to detect small quantities of these macromolecules. these detectors = molecular probes probes help us to find these needles in haystack, they’ll allow us to detect small quantities of RNA or DNA that we might be interested. can use these probes to identify these minute quantites of macromolecules (RNA and DNA) and the way that we can use these tools based on simple principle that RNA and DNA are nucleic acid and in single form they will base pair with complementary sequences. as long as we can make a probe that has a complementary sequence to segment of nucleic acid that we might be interested in, we can detect very small quantities of that particular nucleic acid. way that we might do this is taking complex mixture of protein, RNA and DNA and to find that minute target, best thing to do initially is to reduce the complexity by separating it out on some sort of agarose gel sieve. so we can do agarose gel electrophoresis to separate all these macromolecules out based on size. once youve separated that out, you don’t want to keep it in gel, because these molecules are water soluble and can diffuse. so after going through all this effort to separate these things out, to get to specific sizes, you want to maintain that size. the idea is then to transfer those nucleic acids to a solid phase membrane. a support that will ensure that those molecules are not going to move from that initial position where they migrated to in that agarose gel. . once theyre all on this solid phase support —> membrane, using a probe, we can try to identify our specific needle in that haystack of complexity. introduce probe with this membrane that contains all these nucleic acids, mix it all up, the probe will recognize its complementary sequences and then you get rid of all the probe that was just binding non specifically by doing stringent washes with increase salt concentrations, temperature and detergents. by doing that, you eliminate all this other garbage, these black triangles that have nothing to do with target. and remaining black triangles are the probe molecules that interacted with our target through watson crick base pairing. depending on method used in order to mark probe, detector can take that membrane which is a permanent record of that separation and can actually visualize where nucleic acid are, position on that membrane. critical refer to this as blotting. the membrane is a blot. McGill BIOL200 - Fall 2019 Single stranded oligonucleotides can be labelled using polynucleotide kinase Known sequence that corresponds to a gene product of interest Lys Glu Ala Phe Thr His His Gly 5’-AAG GAG GCA TTT ACC CAT CAT GGC-3’ reverse complement of DNA stretch that were interested in Synthesize an oligonucleotide that has the complementary sequence Polynucleotide Kinase (PNK) will phosphorylate nucleotides by transferring the γ phosphate of ATP to the free hydroxyl at the 5’ end of the oligonucleotide 5’-GCC ATG ATG GGT AAA TGC CTC CTT-3’ P 5’-GCC ATG ATG GGT AAA TGC CTC CTT-3’ making a probe depends on adding on some sort of means of visualizing where that probe ends up, when its interacting with target. one very simple way of synthesizing a probe is to take an oligonucleotide, been synthesized in lab, is complementary to given sequence that we might be interested in. presuming you already know the sequence of interest. shown here a stretch of amino acids and a hypothetical DNA sequence that might eventually be transcribed into RNA that would give rise to amino acid stretch. the complementary © R. Roy, 2019 oligonucleotide probe shown in red. going to interact in complementary manner with that DNA stretch. we have a reverse complement of DNA stretch that were interested in. having an oligonucleotide isnt useful to you unless you can somehow visualize where it is binding to its final target. in order to do that, investigators will mark that oligonucleotide with some sort of radio-labelled isotope, allow it to stand out really obviously and for us to be able to detect it at very low quantities. most common means of doing this is by using enzyme from bacteriophage called PNK (polynucleotide kinase). PNK will transfer the last phosphate (gamma phosphate) on ATP molecule to the 5’ free hydroxyl on oligonucleotide. by carrying out that reaction in presence of isotopically labelled gamma ATP (phosphate last one in chain of 3) that will be transferred to its final target, the 5’ end of that oligonucleotide. by doing this, end up with product thats radioactively labelled on 5’ nucleotide of that oligonucleotide that was synthesized in lab, from that reaction mixture, you have ATP that wasnt used/ incorporated and have the product which is an oligonucleotide thats been phosphorylated by PNK. how would you separate those 2 entities from that reaction mixture? lets say from last few lectures on chromatography, is there one way to use chromatography to separate a small molecule from large molecule ? doesn’t just pertain to proteins. gel filtration, by doing simple gel filtration step, you can get rid of nasty ATP thats radio labelled and end up with oligonucleotide thats labelled on its 5’ end that you can use as probe. gel filtration can be used in number of situations in molecular bio lab, not unique or specific to proteins, can use for macromolecules. helps you to separate things based on size. particularly in preparative context. NEXT SLIDE that way is pretty easy, can make olignocleotide probe, single stranded DNA, end up with great detector that you can use in all reactions. another way you can make probes, generally used for longer probes is by taking advantage of PCR —> 2 primers and introduce new nucleotides that youre adding to reaction during elongation phase or polymerase phase. so, theoretically should be able to add radioactive nucleotides in that polymerization phase. and radiactively label or you can use other means, other types of chemistries, can introduce these labeled nucleotides in the formation of this new segment of DNA, simple PCR reaction. you denature double stranded DNA, introduce primers into the reaction and as you add to dNTP collection, you reduce the amount of one of those nucleotides. usually DCTP. just a little bit, then you add in a radiactively labbelled dCTP. the dCTP has to be labbelled on alpha phosphate. not gamma phsophate like in PNK reaction. because alpha phosphate of dCTP will be incorporated into growing chain, whereas the gamma and beta phosphates are not. by carrying out PCR reaction, everytime that the polymerase has to incorporate CTP or dCTP, it can use either the cold unlabelled dCTP or every once in a while it can choose the radio labbeled dCTP and incorporate that into newly synthesized DNA chain. McGill BIOL200 - Fall 2019 PCR can be used to make labelled DNA probes incorporating dNTPs that carry a radiolabel on the α-phosphate into PCR amplified DNA Unincorporated radioactive dNTPs substrates are removed The radiolabelled PCR product must be rendered single stranded before it can be used + dGTP, dTTP, dATP, dCTP (α−32P dCTP) tried to show here by red C’s. is a doubled stranded DNA probe any good to us in that format? why isnt it good in a double stranded form? not going to bind. you take this probe and boil it, simple, high tech molecular bio. denature that so it can interact through watson crick base pairing with DNA/RNA targets. lazy fast way of making probes GGATTCCA CTTCTGCGTTA……….CGGAGTAGAATTCCGGAAT CCTAAGGTGAAGACGCAAT……….GCCTCATCT TAAGGCCTTA © R. Roy, 2019 McGill BIOL200 - Fall 2019 Analysis of nucleic acids by transferring to membranes (nylon, nitrocellulose) Both DNA and RNA can be separated according to size using an agarose gel. DNA is cut with a restriction enzyme and then run through an agarose gel-a diagnostic signature that reflects aspects of the DNA sequence mRNAs of different sizes will correspond to the various genes that encode them. Both of these analytical techniques require that the molecules be denatured (single stranded) and transferred to a membrane for subsequent analysis. DNA-> nylon, nitrocellulose RNA->nitrocellulose © R. Roy, 2019 these probes are useful when we carry out blotting procedures, described superficially when discussing initial synthesis of probes. can analyze nucleic acids by running them through agarose gels, transfering them to solid phase membranes and then subjecting those solid phase membranes to a hybridization step with the probe that weve synthesized in lab. if youre working with DNA, we refer to this as southern blot —> named after E.M Southern because he was the first person that every thought and actually did the experiment that you can separate DNA out and detect it. following agarose gel electrophoreis on a membrane. if youre going to do southern blot, you prepare DNA sample and then digest it with restriction enzyme. digest it first because if you try to put a big giant DNA molecule into one of these agarose gels, it wont work, wont fit in correctly. absolute mess. but if you cleave it into small fragments or smaller, then can run ffragments through that agarose gel sieve and separate the fragments out according to their size. after youve run the agarose gel for a certain amount of time, to resolve all different sizes, need to render the DNA in the gel into a single stranded form. take gel and expose to alkaline solution, high in NaOH, bathe it around until all that DNA in that gel has become denatured, everything has become single stranded. carefully remove that gel from bath, then introduce it to paper napkin pyramid. what you do is use a property from alkaline solution and use diffusion to direct liquid from the gel towards this solid phase membrane. well shown in diagram. have a bath of alkaline solution on top of that bath, have a platform that will have a piece of filter paper down in solution so it can constantly be in contact with gel, which you place on top of it. on top of the gel, put a membrane could be nylon or nitrocellulose. these things are charged in positive manner so once DNA comes in contact with it, DNA sticks —> wont keep going. on top of membrane, put filter paper and tons of paper napkins. liquid will move from this bath below the gel through the gel and upward into that pyramid of paper napkins. in doing so, it drags along with it the nucleic acids, DNA in this case. when DNA finally gets to membrane, stops and the diffusion keeps going with rest of liquid. the next morning, come back, get rid of paper towels and grab membrane which is thin piece of flimsy stuff. looks like paper. fragile, but on that piece of membrane, you have permanent record of that separation you did on gel. no longer need the gel, throw it out. subject that membrane to UV cross linking step so that the UV will covalently link the DNA to that particular membrane (nylon or nitrocellulose). NEXT SLIDE why would we use a very flammable thing for the membrane? its mostly because its charge and interacts nicely/strongly with nucleic acids. as long as not smoking in lab, okay. but dont want to put stuff near flame because not good. those experiments take place outside lab.get an idea, if clever enough, remember to mark the sizes, where various markers are so you know exactly how to line up radioactive probe with the size of the standards that you ran on electrophoresis gel. you know the size of where that radioactivity is. know the size of that nucleic acid. important because if you do math well and know how many moles of probe you have for amount of radioactivity, can do calculations and figure out how many copies of DNA, youre DNA target might even be on that particular membrane or even in the cells you were working with/samples you started with. important, great technique for quantitative and qualitative analysis. McGill BIOL200 - Fall 2019 Once covalently bound, the levels and positions are permanently recorded The nucleic acids are then bound covalently to the membrane using UV you can then take that membrane and subject it to single stranded probe which youve either boiled before or it could be youre olignucleotide probe that you 5’ end labbelled. you allow those things to get in contact with eachother for a period of time, over night/hours. this step is called hybridization. allow the formation of a hybrid throught W&C base pairing. after hybridization step, remove the probe liquid and then wash that membrane to get rid of all the uneccesary nonspecific radioactivity that could just bind to the positive charges on the membrane itself. This permanently records the levels (abundance) and the position (size) of the molecules following separation on the gel. The support or “blot” can then be “hybridized” with probes to any sequence that may be of by washing really heavily, increasing temperature, end up with membrane thats much cleaner. then you can subject it to autoraadiography in order to visualize the final position of the nucleic acid that youre interested in. because probe is specific to that one Washes remove non-specific signal and only particular target. interest. complementary sequences will be detectable on the blot following autoradiography. © R. Roy, 2019 thisMcGill becomes particularly important when youre starting to do petigrees of either humans or animals. you can come up with BIOL200 - Fall 2019 conclusions about relatedness of individuals in a population by simply examining southern blots of the way that a specific sequence behaves with respect to a given digest with restriction enzyme. Nucleic acid hybridization techniques: DNA detection in this simple data, see that there are 3 different variants that our probe binds to. the three different colours. different colours correspond to where the probe binds to a fragment that seems to be running at a different position in the gel. 1 binds to big nasty fragment. 2 binds to intermediate fragment and 3 binds to a lower molecular fragment. Nucleic acid probes with complementary sequence can bind nucleic acid targets: Southern Analysis (E.M Southern) Molecular identification of Polymorphisms means that theres variation within a population and we can detect that molecularly. with our probe if we do a southern blot. this is just a family pedigree that we can look at up here looking at grandparents, children… and can understand what chromosomes the proginee of this family have based on the detection of these variants. powerful, allows you to determine relatedness between individuals, can also say a lot about diagnostics in certain cases. whether you have or dont have a specific variation in sequence. southern blots are great for this thing, Analysis of DNA-Southern Blot by analyzing the genes to a rough degree, certain regions of genes in individuals in a very fast and cost efficient manner. important if looking at lots of mice, want to see 1 particular gene variant or genotype in population in mice, do a lot of southern blots and find those variants Relatedness and/or Diagnostic © R. Roy, 2019 McGill BIOL200 - Fall 2019 Detecting Polymorphisms with Probes…. Normal Gene Z Probe Ec oR I III nd m Ba Fragment 1 Chromosome Gene Z Hi Y HI Gene X Fragment 2 can use these things over and over. once youve hybridized once with 1 probe, and done with it, can dehybridize it. wash it really stringently and increase temp high so break H bonds and probe lets go. then can reuse it again with another probe that you might be interested in. and when not using it, put in freezer, recyclable technology. we very often use this technology in doing pedigrees, if you want to pedigree something, very often you carry out southern blot. simple example of how we can do that : pretend that this is a choromosome and we know that there are only 3 restriction sites in the middle and happen to fall in gene Z. if we have a probe that corresponds to the DNA sequence, encompassing gene Z, then we have this tool to follow what might be happening in and around the gene Z locus. if we are to cut that chromosome with restricition enzyme like EcoRI, then can see that it would fall into 2 big segments. fragment 1 (big chunk of chromosome) and then there would be a little chunk, fragment 2. both of these fragments would light up with the probe. the probe would recognize the big fragment due to this capacity to hybridize with the big fragment and would also recognze the small fragment due to that small section they can hybridize with. the small section will be less strong than the large section. its a little bit less binding to it, a lot of times it just falls off. © R. Roy, 2019 McGill BIOL200 - Fall 2019 Detecting Polymorphisms with Probes…. Mutant Gene Z Probe Chromosome X Ec oR I nd m Ba III Gene Z Hi Y HI Gene X GAATTC -> GAATCC Fragment 1 if you have a varian in gene Z, mutant gene Z but can be any kind of allelic variant in a population, where a single nucleotide change disrupted the EcoRI site, thats why. put X on EcoRI. single nucleotide change makes it so that the restriction site is no longer recognized by the enzyme. the probe is going to recognize the same region despite that small variation, doesnt care. in this situation, if i try to cut that chromosome with EcoRI, it wont cut anymore and my probe will recognize a big giant fragment, not 2 like in wildtype. but 1 big fragment. polymorphisms, changes in nucleotide sequence that give rise to alterations in the way that restriction enzymes cut up a given sequence. © R. Roy, 2019 McGill BIOL200 - Fall 2019 Detecting Polymorphisms with Pro...
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