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[Music]

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[Applause]

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hi everyone thank you guys for being

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here my name is Tia maka and today I'm

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going to talk to you about some of the

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work we've done concerning the role of

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cyclic phosphates in non enzymatic

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ligation and just studying some general

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principles of non-enzymatic ligation so

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not to belabor the point but we all know

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that non enzymatic nucleic acid

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replication is a complex process and so

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there have been a lot of studies to try

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and understand this and from our point

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of view they're basically two ways in

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which people generally approach this so

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in one case you can look at

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polymerization of nucleotides so you

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have a template and you try and

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polymerize your different nucleotides on

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it but on the second hand if you can

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imagine that there were there was a

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template on the early earth then it's

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not too far to assume that it would also

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be shorter the good nucleotides and so

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the questions we're trying to answer is

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how do you like get the shorter leg of

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nucleotides once you've hybridized them

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together and so we before we started

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this work we looked into literature to

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see some of the things that people had

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been doing because this problem dates

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back to decade old problems and so in

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general like I said earlier there would

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be a template and the idea is you're

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trying to like it won't be a sharp

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primer to your short strands one of the

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features of your ligo's of your of your

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primer is that you would have a

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phosphate and you would need to activate

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this phosphate so people have done this

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by you can have an activated phosphate

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that you then include in your study in

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your application but you can also

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include condensing agents which can then

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activate your phosphate and you can get

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your new strands and some of the things

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we found about condensing agents in

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general was that even though this has

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been studied a lot in the past that did

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not seem to be a consensus on which

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condensing agents were superior so for

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instance we looked at a lot of studies

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with cyanogen bromide which has very

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fast ligation kinetics bots energy

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bromide is highly toxic and the fast

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reaction kinetics actually prevents you

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from understanding the mechanisms on the

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other hand we looked at what else would

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work how would I image and we found that

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it could take weeks and perhaps even

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months for you to get your maximum

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products

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which again is a bit hard to tease out

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the mechanisms involved with ligation

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and perhaps the most interesting thing

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that we found was that generally you had

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lower youth for RNA ligation compared to

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DNA ligation and this is interesting if

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you put it in a context of the RNA world

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and also and perhaps more interesting is

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that a lot of these so if you have an

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RNA or the connector oligonucleotide

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with the three prime phosphate it would

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form a cyclic phosphate which a lot of

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people believe to be an activated

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intermediate and so if you have this

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activated intermediate for the RNA or

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the GU nucleotide why don't you get

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higher yields compared to DNA Lagoon

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acute IDEs and so this brings us to the

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two questions are trying to answer in

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this study the first one is what

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parameters affect the efficacy of

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chemical ligation and the second one is

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how how relevant or how is the cyclic

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does your two prime three prime cyclic

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phosphate actually enhance the rate of a

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chemical equation and to do this this is

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our experimental design so we have a

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hairpin which we've optimized to be very

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stable and we're going to try and like

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get a shot a piece of illegal to the

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stem LIGO is a three prime phosphate and

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generally we mix in our hairpin and our

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leaders together I'm after hybridization

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we add in a EDC so in this case we're

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using EDC what else in the book how

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would i omit as a condensing agent and

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after activation we incubate it for a

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period of time at different temperatures

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and reformed alligator products and we

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typically run our reactions on a gel

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because the hairpin is family bought and

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so that that allows us to see our

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products and so this is just an example

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of so in this study we're looking at the

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effect of the activated nucleotide on

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ligation so our hairpin is all DNA and

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most of the shot oligonucleotides so

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most of the nucleotides on this illegals

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are DNA except for the last one so the

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last nucleotide is either DNA RNA or a 2

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prime or methyl and the idea is that

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once we activate this with EDC the EDC

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attaches itself to the phosphate so

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creating a good leaving group for the

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legations to continue but in the case of

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the irony it forms a cyclic phosphate

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and so again we're looking at the effect

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of this cyclic phosphate on enhancing

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ligation rates and so in this first

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experiments what we do is study at

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different temperatures so this gel I'm

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showing you was taken after only two

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hours so at different temperatures what

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is the effect of having the different

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nucleotides on that terminal end and so

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we see very quickly that for your DNA

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Liga nucleotides as you increase the

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temperature you increase the rate of the

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chemical reaction so this is kind of

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what we expected but you can see that

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after only two hours you're almost at

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80% of chemical ligation products formed

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after at tight temperatures you see that

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for the area nado even though we're from

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in a cyclic phosphate and so we verified

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this with HB s and lc/ms you don't get

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any products formed after two hours

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whereas you get a lot of products formed

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for the DNA but once you block that the

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two prime hydroxyl with an met with a

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methyl so not true prime or my field

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case you see that as you increase the

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temperature you get more products that

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are formed it's also interesting to note

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that at low temperatures so at four

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degrees Celsius you don't see a much of

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any products for mean and we think that

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but we think that this is because at

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this low temperatures the two prime or

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methyl is in it has steric hindrance

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that is preventing this ligation from

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going on and by his not the temperature

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you can reduce the effect of the

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conformation and so we do this via a

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widespread so we do higher temperatures

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but we also look at it for two hours and

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24 hours so the blocks represent two

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hours and the dash represent 24 hours

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and just generally you see that at high

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temperatures for the DNA and the two

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prime or methyl you get about the same

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rate of product or the same amount of

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products that are formed immediately but

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like I showed you earlier and you might

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have we shared it more here after two

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hours the DNA is almost at 60% at 25

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degrees Celsius but a 2 prime or methyl

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is only at 20% and so what this showed

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us was that

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it's important to make sure your

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reaction has gone into completion so if

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we had just looked at instantaneous

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rates you might think that DNA ligation

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was perhaps better than to promote

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methyl but that is very temperature and

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time dependence we also saw that again

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even after 24 hours of four degrees

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Celsius you don't get any products form

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for the to prime or methyl but even on

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the best condition so 25 degrees after

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24 hours you barely see any irony on

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ligation going on now we're not saying

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that you don't get you can get some

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products formed if you increase the

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amount of excess oligonucleotides that

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you have so in this experiment we had

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only 1.5 excess of Olli goes to the

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template but if you increase it to say

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20 times if you saturate the amount of

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illegals that are present then you can

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see some ligation happening if you also

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increase the pH of the reaction you can

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get some products formed but in both

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those cases the maximum product that you

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get is only about 20% whereas are not

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best conditions for DNA are to promote

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methyl you get up to 80 90 percent on

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the product formation and so haven't

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picked the best conformation so we know

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we want to use the DNA oligonucleotide

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primer to do a ligation we decided to

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look at the effect of sequences so this

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has been done before but in our case we

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believe that we had optimized all the

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possible and problems and so we wanted

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to see if they would have any effect on

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our ligation reactions and the things we

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pay attention to here is that this - or

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this line represents the nick of the

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ligation and so in this first gel again

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this experiment was done after only two

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hours but in this first job we're

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looking at the GG being opposite the

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ligation Nick versus being at the

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ligation Nick and again we look at it at

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different temperatures and you see that

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when you're GG so when you have periods

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at opposite the ligation Nick you see a

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really fast increase in the amount of

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products that you get when you increase

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the temperature but when you have the GG

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stocking at the nick of the ligation

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then you see a decrease in the amount of

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products that are formed as you increase

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initially so at 4 degrees Celsius

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there's barely any product forming but

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as you increase the temperature then you

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can increase the reaction rate

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and so again like we signed the case of

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the two primal metal there seems to be a

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difference that there seems to be some

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effect of stacking on our ligation rates

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and so we do this some experiments at

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different times so two hours and 24

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hours studies and we also do we also

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scramble the positions of the GS and the

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C's and you see that generally at high

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temperatures that doesn't seem to be any

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effect on the sequences location but

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after 24 hours when you have your stack

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in at the nick of ligation there's it

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really decreases the amount of products

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that you get compared to any have you're

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stacking opposite the ligation Nick and

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so we decided to look at whether this

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was just a feature of the GG base

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pairing system where this could also be

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applied if we had an 80 base pairing

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system so we kept almost everything the

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same but now instead of CG closing base

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pairs we looked at 80 closing base pairs

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and you see about the same effect that

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we observed so in this first bar when

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you have your purines so you're a a

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stock opposite ligation Nick you say

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really fast increase in the

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instantaneous rate and it approaches

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maximum but when you have your puing

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stocking at the nick of ligation then

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you see a decrease in the amount of

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products that are formed and again as

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you increase the temperature you allow

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it attained more confirmation and you

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can get more products that are formed

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answering conclusion from just a general

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nucleic acid standpoint we see that

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their multiple factors that can affect

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non-enzymatic ligation one of the first

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ones we looked at is just the effect of

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the sugar so having a 2 prime hydroxyl

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versus an O methyl on your

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oligonucleotides we also saw that base

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flanking so the position of the purines

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really affected how fast your reaction

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weren't especially at low temperatures

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and from just an irony award for

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spective we saw our data showed that the

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super imperium cyclic phosphate is

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perhaps not an activated intermediate in

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template director type ligation

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reactions and with this I'd like to

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thank NASA and a cell phone in the

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center my group members are my