Exp11 - Experiment 11 DNA Composition by HPLC1,2...

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1 Experiment 1 1 DNA Composition by HPLC 1,2 Introduction This experiment illustrates quantitative analysis by high performance liquid chromatography and biochemical methods of sample preparation. The sample is calf thymus DNA (Clostridium perfringens) . A suspension of the sample is denatured to give single- stranded DNA by incubation at 100°C for 10 min. The heat-denatured DNA is then digested with Nuclease S 1 , (50°C for 1 hr) to give 5'-mononucleotides (abbreviated C, T, A, and G): In double-stranded DNA, C is hydrogen bonded to G and A is hydrogen bonded to T. Therefore the concentrations of C and G are equal and the concentrations of A and T are equal. DNA from different organisms has different relative amounts of (C + G) and (A + T). When DNA is hydrolyzed by the enzyme nuclease S 1 , it is cleanly broken into the four nucleotides. These nucleotides are separated and quantified by use of a reverse phase HPLC column. Standard solutions of the four nucleotides are used to calibrate the elution times and absorbance signals (260 nm). The peak areas of A, T, G, and C are determined for the standards and for the DNA sample. You will determine the relative concentrations of [A]/[G], [C]/[G], and [T]/[G] and find the fraction of [C + G] nucleotides in this DNA. (1) Wietstock, S. M. J. Chem. Ed. 1995, 72, 950-952. (2) Harris, P.C. "Quantitative Chemical Analysis" 6 th Ed. 2003. w\vw.whfreeman.com/gca.
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2 Background Information 1 Over the years, the most common method utilized for the determination of the combined mole fraction of G + C ( X gc ) is the melting temperature (T m ) of the DNA as determined by a spectrophotometric method (1-3). This involves observing the 260 nm hyperchromic shift of the DNA as it is transformed from double stranded DNA to single stranded DNA (denaturing the DNA greatly increases the absorbance at this wavelength in comparison to the double stranded DNA). The DNA in this assay is suspended at a given concentration in a buffer system of known ionic strength, typically a 0.05-0.15 M sodium chloride-sodium citrate buffer solution. This sample is placed in a spectrophometer with a water-jacketed cell holder, and a micro- temperature probe, connected to a recording system, is placed in the sample. The DNA suspension is heated slowly while the absorbance at 260 nm (A 260 ) is recorded. A plot of the A 260 versus incubation temperature is prepared and the T m of the DNA is read at the midpoint of the hyperchromic shift. If the salt concentration and the T m of the DNA solution are known, then the X gc can be determined utilizing the equation: T m = 16.6(log C s ) + 41 X gc 81.5 where C s is the total salt concentration of the sample (1). Determining X gc by this method is not a trivial task when dealing with X gc greater than 0.6 because the T m occurs near 95°C. A new method for the determination of
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This note was uploaded on 02/16/2012 for the course CHEM 125 taught by Professor Na during the Fall '11 term at Purdue University-West Lafayette.

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Exp11 - Experiment 11 DNA Composition by HPLC1,2...

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