Question

You have sequenced a fragment of DNA and produced the gel shown in Figure Q10-50. Near the top of the gel, there is a section where there are bands in all four lanes (indicated by the arrow). Which of the following mishaps would account for this phenomenon? Explain your answer.

(a) You mistakenly added all four dideoxynucleotides to one of the reactions.
(b) You forgot to add deoxynucleotides to the reactions.
(c) Your primer hybridizes to more than one area of the fragment of DNA you are sequencing.
(d) A restriction nuclease cut a fraction of the DNA you are sequencing.

Answer 1

Bands in all four lanes of a DNA sequencing gel suggest that all four dideoxynucleotides were added to one of the reactions by mistake, causing DNA synthesis termination at the same base across all reactions.

Step-by-step explanation:

If you observe bands in all four lanes near the top of a gel during DNA sequencing, it indicates that sequences have been terminated at the same point in all four reactions.

The likelihood is that you mistakenly added all four dideoxynucleotides (ddNTPs) to one of the reactions, resulting in the termination of DNA synthesis at the same base for each ddNTP.

This mishap leads to bands appearing in all lanes at the same height, as each combination of DNA polymerase, one type of ddNTP, and the template would have stopped at the same point.

It is important to remember that in Sanger sequencing, each reaction ideally contains only one of the four ddNTPs alongside the regular deoxynucleotides (dNTPs).

Each ddNTP is fluorescently labeled, so when they terminate DNA synthesis, they can be identified by their distinct fluorescence during gel electrophoresis.