How can i learn to read and compare western, northern, and southern blots? genetics test coming up =)?

Question

also would like information on after reading blots, what mutations may cause the blot results (i.e. 5'UTR, missense, frameshift, etc.) Thank you so much!!!

Answer 1

You know that you are pretty much asking for someone to write a whole chapter of a molecular biology textbook, right?  But I'll give it a try ...

Southerns are DNA blots (named for Ed Southern = should be capitalized), northerns' are RNA, and westerns are protein blots.  In each case, a gel is run, then transfered to a membrane, and then probed.  The probes are DNA for DNA and RNA blots, and antibodies for westerns, and are somehow rendered detectable (there are many ways to do this).  In each case there will be size standards to tell you what size the band is.  DNA will be reported in base pairs/bp, RNA in nucleotides/nt, and proteins in kilodaltons/kd.

To learn to read these blots, you just need to look at them and get acquainted with the format.  Search the biomed literature at the link and find papers with blots, look at the pictures, and read the captions, and use that info to understand the data.

As for the mutations ...

Since Southerns detect DNA via base pairing hybridization, they will not usually pick up point mutations.  They can sometimes detect larger DNA rearrangements, and this is seen with altered band sizes or missing bands on the blot.

Since northerns detect RNA, the ability to detect a band depends on production of enough stable RNA to be detectable and on the production of an RNA with bases complimentary to the probe.  Thus, it becomes a case by case situation.  A large rearrangement might block production of the RNA, decrease stability of it, or remove the bases to which the probe would bind, but it might also allow production of an aberant RNA that still hybridizes - and this could be the normal size or it might be larger or smaller.  A promoter mutation might increase or decrease production of the RNA, or might alter induction in some specific situation, but the size would be normal.  A splice site mutation might alter RNA abundance, or might change the size, or do both.  Coding sequence point mutations wouldn't be detectable directly by a northern, and missense mutations would not be typically detectable by northerns at all, but frameshifts might alter the abundance of RNAs by nonsense mediated decay - although this is dependent on the location of the mutation relative to the gene's exons.  As I said, the effect on a northern all depends on the exact nature of the mutation.

Westerns rely on antibodies.  If the mutation disrupts the antigen(s) the antibody binds to or if it blocks expression of any protein, then there will be no band.  On the other hand, if the mutation doesn't alter the expression of the antigen, then the protein band might be larger, smaller, or the same on a blot.  It all depends on the precise natures of the mutation and of the antibody.

Hope this helps.