bioc441 - 2008AnsProbSet1a

bioc441 - 2008AnsProbSet1a - Biochem 441 Ted Young Page1/4...

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Biochem 441 Answers to problem set #1a Ted Young Page1/4 1. DNA is usually denatured in ‘2X SSC” (SSC is 0.15 M NaCl, 0.015M NaCitrate, the latter serving as a buffer; a stock solution of 20XSSC is usually diluted 10X for this purpose). Bacteriophage lambda DNA has Tm of 85 o in 2XSSC. If you accidentally diluted the stock buffer 100X would the Tm change? If so, in what direction? Why does this happen? Yes. It would decrease. Moderate ionic strength is needed to shield the negatively charged phosphates. At low ionic strength ionic repulsion decreases the stability of the DNA and lowers the Tm. Wildtype phage DNA has a 52%(G+C) content. Some mutants of the phage have lost a segment of their genome that is 80% (G+C). Would their TM be different? If so, would it be lower or higher? The Tm would decrease because the (A+T) content would be higher in the mutants. Tm is directly related to (G+C) content. In the electron microscope DNA can be seen as a thick filament after it is coated with protein and electron-dense atoms such as Pt or Pd. The two strands aren’t resolved; a single dark line of duplex DNA is observed. Single-stranded DNA is visualized as a lighter line When a mixture of wildtype and mutant phage DNA is mixed, heated above their Tm’s, and allowed to renature by slowly lowering the temperature to 25o, heteroduplexes form that contain one strand of mutant and one strand of wildtype DNA. In addition homoduplexes also arise. These three types of DNA can be distinguished by studying their length and configuration in the EM. In the figure below label wildtype (wt) and mutant (m) homoduplexes and heteroduplexes (het). What are the loops of DNA emanating from the two unusual forms? What does the base of the thin loop represent? How could you test this idea using the three types of enzymes listed below. a. Nucleases that non-specifically cleave double-stranded DNA (meaning they cut between any bp). b. Restriction enzymes. c. Nucleases that non-specifically cleave single-stranded DNA.
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Answers- problem set #1 Page 2/4 Problem #1 continued Answers. WT=long lines Mutant = short lines Heteroduplexes = lines with a single-stranded loop. The loops or thin lines are single-stranded DNA representing the region deleted
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bioc441 - 2008AnsProbSet1a - Biochem 441 Ted Young Page1/4...

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