Posts: 3,675,937
Threads: 734,344
Joined:
Sep 2021
Reputation:
5
A laboratory worker notes that when she tries to hybridize a radioactive probe (10 nucleotides long) to a Southern blot of DNA from cultured cells, a solid, dark pattern emerges. What is the most likely reason for this pattern?
A. The temperature of the hybridization mix is too low, resulting in aberrant signals
B. The salt concentration of the mix needs to be increased
C. The cell culture is contaminated with mycoplasma
D. A longer probe is needed to increase specificity
E. The blot contains incompletely digested DNA and hence the complementary sites are not adequately separated
Posts: 3,675,937
Threads: 734,344
Joined:
Sep 2021
Reputation:
5
hi, could someone please explain this answer. thanks
Posts: 3,675,937
Threads: 734,344
Joined:
Sep 2021
Reputation:
5
The answer is D. A probe of only 10 nucleotides (even if it does not contain an obviously
repetitive sequence) will have over 1000 matches in the genome. Raising the temperature of the hybridization mix may improve the signal but eliminating competition from ∼1000 sites is unlikely (choice A). Increasing the salt concentration also can increase stringency but is not
likely to eliminate the overwhelming number of competitive sites (choice B). Although
the cell culture might be contaminated, unless the probe is related to the contaminating
sequence(s) this will not be the problem (choice C). Incompletely digested DNA on
the blot can lead to aberrant hybridization, but using a short probe is likely to give a
broad smear of signals (choice E).
Posts: 3,675,937
Threads: 734,344
Joined:
Sep 2021
Reputation:
5
the probe is very short, and will hybridize any region with the same/almost similar chain of nucleotides, and therefore it bind to a LOT of areas in the whole DNA. If the probe were longer, it would have more specificity, beacuse the longer the sequence gets, the lesser the chance that the very same/similar sequence would be present in other areas.
