You have just been employed by a small biotech company that, among other things,

Question

You have just been employed by a small biotech company that, among other things, sequences DNA for individual labs. Your job is to set-up and run the experiments. The first set of reactions you set up fails. All of the reactions stopped within the 1st several bases despite the fact that the reads should have been hundreds of bases long. The individual training you set up some reactions and ran them in parallel to yours and of course all of her reactions were a success. Apparently you have made a rookie mistake and upon inspection of your six reactions it becomes immediately obvious what happened. Here are the data produced from those six reactions. From these data can you determine what your mistake was? How do you know? By the way, each sequence represents what was attached to the primer, which has been omitted from these data (10 points). a. 5- ATTGC-3

b. 5- AC-3

c. 5-C-3

d. 5- GGATTATGGC-3

e. 5- GATC-3

f. 5- TTAGGAC-3

Explanation / Answer

Answer:

From the data, all the six reactions are getting terminated at Cytosine. In a typical reaction set-up the concentration of fluorescent tag labeled dideoxynucleotides triphoshphates (dNTPs: dGTP, dATP, dTTP and dCTP) is always in excess. The dideoxynucleotides (ddNTPs) are also added to the reaction mixture. Since ddNTPs lack the 3'-hydroxyl group that is required for DNA elongation therefore whenever these are incorporated in the DNA strand DNA synthesis will stop, chain termination will occur and it will not extend further. Each reaction tube contains a different ddNTP (ddGTP, ddATP, ddCTP or ddTTP).

Since all the reactions are terminating at cytosine and the length of the sequences obtained is only several base pairs vs the expected size, therefore one possibility could be that in the experiment, instead of dCTPs only ddCTPs are added and that too at higher concentration leading to termination of the reaction at cytosine whenever a ddCTP is incorporated and therefore smaller sequences are obtained.

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