Question

During DNA sequencing, why is the autoradiogram read from bottom to top to arrive at the original sequence ?

Hint:

Remember how DNA fragments are separated on the basis of size and the different properties of the vectors.

Answer 1

(i) Reading Autoradiogram from Bottom to Top: During DNA sequencing, the autoradiogram which is obtained after gel electrophoresis is read from bottom to top to determine the sequence. This is because the smallest fragments migrate fastest and reach the bottom of the gel and the larger fragments move slowly and stay at the top. As DNA synthesis occurs in 5' to 3' direction, reading from bottom to top shows the 5' to 3' sequence. (ii) Single-tube DNA sequencing considered better and safer: Single tube sequencing is considered better and safer because it is automated, faster, and uses a single lane for gel electrophoresis which simplifies the process. It also uses fluorescent dyes, which are safer compared to radioactive isotopes that are used in the original method. The results can be directly displayed on a computer screen, and it is easier to store the data. (iii) Vector Preference for Single-Stranded DNA Sequencing: M13-based vector will be the preferred vector for sequencing single stranded DNA. This vector carries single stranded DNA within the viral particles, which makes it ideal for cloning single-stranded DNA.