Question: Copy #: 0 Name: Sequencing Question: 10 Points Total A. You are doing Sanger dideoxy sequencing using a gel. On the next page is the gel with the ddNTP+dNTP mixtures labeled. Draw in all the bands of DNA (e.g., as if you used ethidium bromide to visualize DNA). (Note that there will be (i.e., you should show), for instance, unincorporated primer. Assume that unincorporated ddNTPs and dNTPs have run off the gel.) The wells are at the top. Beside the "gel" are the number of bases, including the primer when applicable, for bands at that horizontal level. (Assume that it is no longer double-stranded when it is run in the gel.) Before you draw the bands, you may wish to look over the rest of this question - especially the last sentence; there is more information to be included than simply "a band shows up here with ethidium bromide". This part alone is 2.5 points. B. Label in the gel: 1. The bands that you would see if, instead of using ethidium bromide, you tagged the ddNTPs (e.g., with a fluorescent dye). Label these bands with a "1". 2. The bands that you would see if, instead of using ethidium bromide, you tagged the dNTPs (the single nucleotides - not the primers) being used for synthesis (e.g., with 32P). Label these bands with a "2". 3. All the bands of DNA (e.g., what you could see with ethidium bromide) if you left out the components in parentheses below. Label these bands with a "3". (Hint: Remember the last question on the sequencing lab?) Bands can have multiple numbers. You may wish to draw the bands as something like "--123--" (without the numbers that aren't applicable for the band in question, of course). Copy #: 0 Name: Below are the sequences of the primer and a template strand of DNA that you are trying to sequence. The primer is, as you can see, to the first 4 nucleotides; the template strand is 18 bases long. Primer: TAGA Template: ATCTACCCTTTGGGTAAC Here is the gel with the mixtures for each lane (the parentheses are for question 3 of part B): Tube #1: Tube #2: Tube #3: Tube #4: ddTTP ddATP ddGTP ddCTP (dTTP) (dATP) (dGTP) (dCTP) dATP dTTP dTTP dTTP dGTP dGTP dATP dATP dCTP dCTP dCTP dGTP bases -------------------------------------------------------------------- | Well | Well | Well | Well | |---------------|---------------|---------------|---------------| | | | | | 18 | | | | | ----------------------------------------------------------------- | | | | | 17 | | | | | ----------------------------------------------------------------- | | | | | 16 | | | | | ----------------------------------------------------------------- | | | | | 15 | | | | | ----------------------------------------------------------------- | | | | | 14 | | | | | ----------------------------------------------------------------- | | | | | 13 | | | | | ----------------------------------------------------------------- | | | | | 12 | | | | | ----------------------------------------------------------------- | | | | | 11 | | | | | ----------------------------------------------------------------- | | | | | 10 | | | | | ----------------------------------------------------------------- | | | | | 9 | | | | | ----------------------------------------------------------------- | | | | | 8 | | | | | ----------------------------------------------------------------- | | | | | 7 | | | | | ----------------------------------------------------------------- | | | | | 6 | | | | | ----------------------------------------------------------------- | | | | | 5 | | | | | ----------------------------------------------------------------- | | | | | 4 | | | | | ----------------------------------------------------------------- Master: Copy #: 0 Primer: TAGA Template: ATCTACCCTTTGGGTAAC Tube #1: Tube #2: Tube #3: Tube #4: ddTTP ddATP ddGTP ddCTP dTTP dATP dGTP dCTP dATP dTTP dTTP dTTP dGTP dGTP dATP dATP dCTP dCTP dCTP dGTP bases -------------------------------------------------------------------- | Well | Well | Well | Well | | | | | | | --23-- | --23-- | --123-- | --23-- | 18 | | | | | | --12-- | | | | 17 | | | | | | --12-- | | | | 16 | | | | | | | --12-- | | | 15 | | | | | | | | | --12-- | 14 | | | | | | | | | --12-- | 13 | | | | | | | | | --123-- | 12 | | | | | | | --12-- | | | 11 | | | | | | | --12-- | | | 10 | | | | | | | --123-- | | | 9 | | | | | | | | --12-- | | 8 | | | | | | | | --12-- | | 7 | | | | | | | | --123-- | | 6 | | | | | | --13-- | | | | 5 | | | | | | --3-- | --3-- | --3-- | --3-- | 4 | | | | | ----------------------------------------------------------------- [2.5 points for EtBr-shown bands: -0.5: Wrong direction, plus will include wrong complementation (-1 total). -0.5: Wrong complementation -0.5: Otherwise incorrect bands for main part -0.5: Forgets primer bands. -0.5: Forgets template bands.] B. Label in the above (7.5 points total this part): 1. The bands that you would see if, instead of using ethidium bromide, you tagged the ddNTPs (with a fluorescent dye, we'll assume). Label these bands with a "1". [1.5 points; -1 if put in on primers or on inappropriate template (-0.5 for one, -1 for both) - don't count as "right" (or "wrong") if have forgotten primers or template(s); -0.5 if problems with main bands.] 2. The bands that you would see if, instead of using ethidium bromide, you tagged the dNTPs being used for synthesis (e.g., with 32P (radioactive phosphorus)). Label these bands with a "2". [3.5 points; -.5 if put in on length-5; -1 if put in on primers - don't count as "right" (or "wrong") if have forgotten primers; -.5 if not in on all four template bands (allowing for lack of said bands); -1 for failure to put in on main ones; -1.5 (including failure to put in on main bands) if put in bands that aren't there at all] 3. The bands that you would see if the DNA polymerase always used a ddNTP instead of a dNTP if possible, and you were using ethidium bromide. Label these bands with a "3". (Hint: Remember the last question on the sequencing lab?) [2.5: 1 for middle, 1 for forgetting either primer or template, or 1.5 for both forgotten; remember may have already taken off for primer and/or template above, in which case middle counts more. Getting at least one right on the middle counts as right for at least half of it (as in if people put in a 3 on the last middle band, but not the other two).] Bands can have multiple numbers. You may wish to draw the bands as something like "--123--" (without the numbers that aren't applicable for the band in question, of course).