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Lecture 17

BIO240 Lecture 17

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Jennifer Harris

Lecture 5: DNA Sequencing Lecture Outline: 1) Dideoxy chain termination method • Manual • Automated 2) Genome databases 3) Blotting Readings: Alberts textbook, Ch 8, pp. 538-540, 548-550 Which of statements regarding DNA replication is correct? a) No enzyme that catalyzes 3’ to 5’ nucleotide polymerization has ever been found. TRUE b) DNA is conservative process (semiconservative) c) Helicase never associates with leading strand (do occur on leading strand) d) Polymerase adds dideoxynucleotides onto nascent strand using energy of hydrolysis of terminal 2 phosphates (Dideoxynucleotides are not added – deoxynucleotides dNTPs are added) e) Okazaki fragments on lagging strand are composed of DNA fragments (composed of RNA primer & DNA – not only DNA fragments) Why sequence DNA? 1) Difficulties with protein sequencing: lots needed, less stable (than DNA), code degenerate (several combos of nucleic acids – more than 1 DNA sequence can actually code for same protein sequence) 2) Protein sequence can be inferred from DNA sequence Dideoxy (Sanger) method of DNA sequencing Ingredients: Steps: 1) polymerase 1) Anneal primer 2) dNTPs (nucleotides – basic building blocks of DNA) 2) Extension by DNA polymerase (to chains of 3) ddNTPs (dideoxyribonculeotides – nucleotide terminator varying lengths) that when incorporated will actually halt synthesis) 3) Run gel (separate out those chains of varying 4) primer (polymerase must add onto to something – initial lengths) primer that polymerase will anneal & can extend from) - In an in vitro setting (lab), can use DNA polymerase to extend, from template, nascent chains of varying lengths. Chain terminators: ddNTPs Types of nucleotide: dNTP ddNTP – Exactly same except they lack 3’ -OH group (does not allow nucleic acid chain to elongate any further b/c polymerase requires that for further extension for formation of phosphodiester bond) - Blue is primer – designed primer so it is complementary to template. Note * means radioactive. - In test tube, have template DNA, primers, dNTPs & ddNTPs. - Do reaction in 4 test tubes, run it out on gel – gel separates fragments based on size – smaller fragments move faster. - 1 thing is to anneal primer & then you allow polymerase to do its work in presence of all those ingredients mentioned previously – allow polymerase to elongate. Up until last C, it was incorporating regular nucleotides (dNTPs) & then for last 1, it incorporated ddNTP so it stops there. In this reaction, there is both ddCTPs & regular dCTPs – both kinds in reaction: kind that allows for chain elongation as well as kind that stops it. - Need different tubes for each 1 of nucleotides (ddATP along with all regular nucleotides – A, C, G, T, etc). Allow this reaction to proceed in all of these different tubes – run each tube out on gel in different lane – keep them separate b/c under traditional method of dideoxy sequencing where label (can be on primer or nucleotide) is radioactive label – keep reactions separate b/c it’s only way to know which nucleotide it terminated on. - When you run it on gel, all of shorter fragments run faster – need gel that can actually distinguish b/w fragments of 1 base in size that differ so we can know exactly where we are in the template – read up from bottom – C, T, G, A, C. - Here you have template sequence – actually double stranded sequence even though in any 1 particular reaction, actually sequencing ssDNA. - Denature strand & anneal labeled primer which determines which of strands you’re going to sequence & which part of strand you’re going to sequence. st - In 4 different tubes, have all nucleotides in excess, in 1 tubes have 1 of ddNTP (A, T, C, G). - In this tube all of fragments are those that end in A – some probability of terminating synthesis there. DNA sequencing vs. replication (DNA sequencing is what occurs in lab via this protocol, replication is what actually occurs in cell) 1) DNA primer used (in DNA sequencing in lab) instead of RNA primer – DNA primers are more stable than RNA primers – less susceptible to degradation – also no requirement to use RNA b/c we’re not going to replace those sequences later on). 2) Heat used instead of enzymes to separate strands 3) Is there a replication fork? No 4) Why? Strands completely separated (denatured) - Disadvantages of DNA sequencing: if you have 2 entirely separated single-stranded molecules, can form all sort of kinks/re-anneal to each other & there are no SSB proteins around to stabilize ssDNA. 1 of ways to get around this in lab is to use polymerases that are active at higher temps – kinks & H-bonds don’t form as much at higher temps. Another disadvantage is that polymerase doesn’t have all those accessories – sliding clamp that helps to hold DNA polymerase onto DNA strand is not there – what happens is that DNA polymerase tends to fall off DNA template much more than in DNA replication – can only sequence relatively short fragments (up to several hundred nucleotides in length – 800). - Advantages: can do it easily in lab & only few ingredients are needed – in vivo there are all these proteins that are required. DNA sequencing review questions 1) Why is ratio of ddNTPs low compared to dNTPs? If you increase ratio of ddNTPs, you increase chances that you will terminate synthesis at every step in which that particular base is required. If ratio is too high, synthesis going to terminate at shorter fragments & you’re going to sequence only 10 bases. If you have too little ddNTPs, tend to only get longer fragments. 2) Why do you read gel from bottom? Smaller fragments run to bottom of gel. 3) Why must your gel be able to resolve differences in DNA fragment sizes of only 1 base? Otherwise, sequence you read from gel will be ambiguous. 4) Why must 4 sequencing reactions be loaded in different lanes of gel? Each 1 of 4 sequencing reactions is terminating with different dideoxynucleotide & you have to keep those separate otherwise you will not know identity of particular band in gel you are reading. 5) Why add only 1 type of ddNTP per reaction tube? Need to know that everything in that particular tube is terminating on 1 type of nucleotide only otherwise results will be ambiguous. 6) Why are ddNTPs not found in cells? Would not be able to replicate any of your DNA – terminating all DNA replication. 7) What would happen if you added 2 types of ddNTP to 1 tube? All of fragments contained in tube could have terminated with 1 nucleotide or other nucleotide. Based on results on running single tube out on gel, would not be able to tell difference. Manual (old-fashioned) vs. automated (newfangled) sequencing - Old-fashioned way was to do radioactive sequencing: have someone in lab pipetting away, setting up reactions, loading reactions onto sequencing gel, reading gels, doing base calling. Improvements in automated sequencing? 1) Instead of radioactivity (to detect sizes of fragments), ddNTP’s are labeled with fluorescent dyes – can have different coloured dye attached to different types of nucleotides – can run all of reactions with dideoxynucleotides in same test tube. 2) Only reaction tube needed 1, not 4 3
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