Question
For the most part I understand Sanger's method and even how topredict the bands that will show up on the gel, but for thisquestion below there is an added "trick" that I cannot figureout.
Question:
You enzymatically elongate primer 5’-[32P] CTG AAA AGG TGGCAT CAA TT in complex
with the DNA template (TACTA CTATTAGTG AATTGATGCC ACCTTTTCAG) using three dNTPs (dATP, dCTP and dTTP) in the presence of one of theddNTPs.
Predict the band pattern in the gel after separation of fourreaction mixtures containing one of the four different ddNTPs.
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I'm not sure what sequence to draw the bands out for? Theentire primer or the template?
Explanation / Answer
the whole template because you are elongating the primeraccording to the template. now the first fragment might have to be the primer itself.....then build upon that. you have a different ddNTP in each tube, so you're only goingto get fragments that end with the corresponding ddNTP.... thedNTPs elongate normally. now i don't want to confuse you but in order to do Sanger'syou actually should have all 4 dNTPs in the tube SPIKED with oneddNTP..... that way, some of the fragments will continue to groweven after adding the nitrogenous base corresponding to the ddNTPof that tube. if you only have the fourth NTP in the form of ddNTP,elongation cannot proceed after the first one is added because itis di-deoxy, so, if the question really does ask that you only have 3 dNTPand the fourth being a ddNTP, then you will only have one band perlane (tube) do you see what i'm saying?