Question

Which of the following statements about manual Sanger sequencing is true? View Available Hint(s) Which of the following statements about manual Sanger sequencing is true? Each of the four terminating ddNTPs is labeled with a different fluorescent dye. The DNA sequence is read from the top of the gel to the bottom. One sequencing reaction is performed.

Answer 1

Answer:

One sequencing reaction is performed

Explanation:

The manual Sanger sequencing technique is based on the termination of DNA synthesis by the addition of a ddNTP, which impairs the full elongation of the molecule. Originally, you would perform four parallel reaction, one for each type of ddNTP (A, G, T, C). So in each reaction you would get sequences of different lengths, all finished with the ddNTP you added in that tube.

By running an electrophoresis for nucleotides, using a gel with enough resolution to separate the sequences by on base pair (bp). If you run each of the reaction mixture in a different rail, you should get fragments of the lengths corresponding to all the positions the nucleotide you added as a ddNTP occupy in the sequence.

Also, as the fragments are separated based on their molecular weight, so smaller ones migrate further in the gel. That means the gel should be read from bottom to top (note that the smaller fragments were terminated earlier than the larger ones).

For example, if in the rail of ddATP, you get a fragment of four bp, that means the fourth nucleotide of the sequence it's an A.

Per each reaction, only one sequence can be sequenced, otherwise it would be impossible to know which fragments correspond to the different sequences.

This method doesn't need any kind of dye to be used on the different ddNTPs as long as they are added in separate reactions.