11_DNA%20analysis

11_DNA analysis - Thermodynamic Analysis of DNA/RNA(Determination of Tm DNA Four different bases adenine(A guanine(G thymine(T cytosine(C base

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1 adenine (A) guanine (G) thymine (T) cytosine (C) Four different bases: DNA adenine (A) guanine (G) uracil (U) cytosine (C) RNA base pairs: A – T A – U G – C G – C Watson-Crick base pairs Thermodynamic Analysis of DNA/RNA (Determination of T m ) Watson-Crick base pairs in DNA A – T G – C [G] = [C] and [A] = [T] Note : Other base pairs can also be formed, but they are less energetically favorable. Example: G 0 (kcal/mol) C – C -1.97 G – G -4.1 to -5.4 C – G -5.4 to -6.8 Melting and Stability of Double Strand DNA • DNA melting = unwinding of the double helix • One way to study the stability of DNA is to determine its thermal stability. (In cells, DNA melting is mediated by specific proteins.) • Method to determine T m : [DNA] measurement via UV absorption at 260 nm 260 wavelength Absorbance single stranded DNA double stranded DNA • The thermal stability is characterized by the melting temperature, T m .
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2 GC pair is more stable than AT pair (since there are 3 hydrogen bonds between GC and 2 bonds between AT) T m T at which ½ of the DNA are double strand and the rest are single strand. [GC] in the DNA strand increases Temperature ( 0 C) 65 75 85 Absorbance 1 1.4 T m = 77 o C if [GC] = 20% T m = 100 o C if [GC] = 78% T m from organisms with different [GC] The equations relating T m and GC content will be given later. If the melting of DNA double strand can be considered as a single step process , i.e., all nucleotide pairs are separated simultaneously, then D S 1 + S 2 ] D [ ] S [ ln RT G ] D [ ] S ][ S [ ln RT G G 1 2 1 2 0 0 For self-complementary sequences, S 1 = S 2 . (since [S 1 ] = [S 2 ]) • The fraction of single strands that remain in the double strand at equilibrium , ts c ] D [ f 2 D c ts = 2[D] + [S 1 ] + [S 2 ] (for non-complementary sequences) c ts = 2[D] + [S] (for self-complementary sequences) (total single strand concentration) (note: f D
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This note was uploaded on 01/17/2010 for the course BME 100 taught by Professor Yuan during the Fall '07 term at Duke.

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11_DNA analysis - Thermodynamic Analysis of DNA/RNA(Determination of Tm DNA Four different bases adenine(A guanine(G thymine(T cytosine(C base

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