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Topic 28

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Western University
Biochemistry 2280A
Eric Ball

Topic 28If you have dna duplex and u heat it or put it in alkaline it will denature The two strands will come apart The two strands will melt any one DNAcharacteristic of how it melts is dependent on dna sequence Melting temperature TMgiven any specific dna at concentration of 1 molar the temperature at which 50 of the molecules are single stranded is defined as melting ptConc of Dnawere not talking about one moleculein population of moleculehalf of them meltednot one being half melted Pattern here given generically Given specific dna at concentration of 1M the temperature at which 50 if the molecules are single stranded is defiend as the melting temperature TMPattern is shown hereat low temp dna is double stranded As you heat up molecule melts and at the temperature half of them are melted TM and as you go higher all of them are meltedWhere half of them is melted TM What determines melting characters that determine melting temp some factors are extrinsic env that dna is in and intrinsic characteristic of dna itselfFirst factor intrinsic Intrinsic features of molecule itself 1GC content Dna wit more GC content will have higher melting temp its cuz they have more H bonds and also has more base stackingthe melting curve is showna regular dna is 5050 gc and at will occur in the middleif solely gcit will be shifted to right higher tmand one entirely at will be shifted to left and have lower TM More gc content higher the melting temp Length of DNA The longer the dna the more hydrogen bonds you have the more base stacking you have therefore you have higher melting temp length of dna is huge factor in determining TM Putting length and GC content together u can get rough formula TM4degrees C for each GC2 degrees C for each AT The longer dna higher tm and more gc higher tmDegree of complementarily between the two nucleic acid strands Exact matches are best but some mismatch can be tolerated The bottom has mismatch at one base position This will form duplex To form duplex you dont have to be exact match But TM drops significantly if theres a mismatch You can form duplexes where there are mismatchesbottomC and Amismatch at one basethese two will form duplexto form duplex you dont have to have exact match but TM drops significantly if there is a mismatch
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