NBME Answers : Block 1 : #9 = During an experiment, a Southern blot analysis is ...

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During an experiment, a Southern blot ...
Gene rearrangement🔍

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submitted by sugaplum(153),

This question is asking about VDJ rearrangement which happens in the bone marrow. The genes are all chopped up because the B cell is trying to generate a unique combination for its receptor
simple concepts... odd wording

Chapter 3 of "how the immune system works" - awesome book

varunmehru in the question stem, they are asking about a constant region. VDJ rearrangement is for the variable. It doesn't make sense :( +1

sallz Both the constant (heavy chains) and the light chains undergo gene rearrangement. The heavy chain undergoes V(D)J random recombinations, while the light chain undergo VJ random recombinations. So gene rearrangement could work for both regions. +5

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submitted by em_goldman(13),

I believe (pls correct me if I'm wrong) you would have similar Southern blot results seen in B cells undergoing somatic hypermutation, but that takes place in the secondary lymphoid tissue, not in the bone marrow.

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submitted by waterloo(19),

You can look at this question strategically.

Southern blot --> so DNA.
they are using an endonuclease to transfer bits, this doesn't really mean anything to you yet.
cDNA is probing for single immunoglobulin-constant region.

That's important, because now you know they are looking at the constant region of immunoglobulin aka Fc of the heavy chain.

So why is this cDNA that encodes Fc, lighting up only once in other tissues, but the same cDNA lights up multiple times in lane 2 (bone marrow)? This is because, at the bone marrow random recombination of light or heavy chain occurs, and our cDNA is present in different random combinations with light chains. That specific cDNA sequence they used didn't change between the tissues or even within the bone marrow, and it will only bind to the one gene sequence. The fact it's lighting up multiple times tells you that the same sequence is present, your question is why so many times.

Also a side note, southern blots are time consuming, so labs use other methods to do the same thing (like PCR), but southern blots are still the best when it comes to checking immunoglobulin and T cell receptor gene rearrangement.

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submitted by benwhite_dotcom(226),

Southern blots are commonly used in immunological studies, as the southern blot allows for the study of DNA alterations. What is normally one gene configuration related to immune globulins in most tissues demonstrates multiple different bands in the bone marrow, indicative of gene rearrangement. This is basically how we create new antibodies. Reactive processes are polyclonal (multiple bands); leukemia, in contrast, is monoclonal (single band).

ali I still don’t understand this one. Could you provide a better explanation? +

benwhite_dotcom The cDNA tag is tagging a constant region common to immunoglobulins, so it normally only finds the one band corresponding to that particular gene (the bands travel different amounts due to their differing size/weight). In the bone marrow sample, that gene has rearranged itself, so the cDNA clone instead tags multiple different genes that are of different sizes on the gel (each one has that same constant region the cDNA is tagging, but with different stuff around it such that the restriction enzyme has cut it up differently). I’d be happy for someone to step in and do a better job on that explanation. +5

em_goldman A Southern blot starts by cutting DNA strands at a particular (short) site and running them through gel electrophoresis, so identical DNA sequences get cut at the same site and thus are the same length, so they are at the same place on the gel. If there's lots of different sequences, the restriction endonuclease (the scissors) cut the DNA at different places, leading to strands that were the same length originally but are now lots of different lengths -> different places on the gel. But how do you know this is the same gene, just with different mutations? The Southern blot uses a probe to look for a more specific (long) region of DNA that you know is in the target gene. So even though there are mutations causing the less-specific endonuclease to cut the DNA at different parts, the overall architecture of the gene is similar enough that the probe can bind, thus we know it's the same gene. (And in bone marrow WBCs, the mechanism here is genetic rearrangement.) +