We saw that Southern blotting of DNA from the ?A allele produces two DNA bands corresponding to fragment lengths of 1150 bp and 200 bp based on the location of two DdeI sites

Question

In contrast, Southern blot analysis of ?S-allele DNA produces a single DNA restriction fragment, measuring 1350 bp in length, since one of the DdeI sites is altered by SNP variation. Why is Southern blotting necessary to identify the correct bands–if you know the sizes, why can't you just identify your band on a gel? Why is the location of the probe (spanning the predicted lost DdeI site) important?

Answer 1

In a Southern blot, you are digesting genomic DNA, and the restriction digest may yield hundreds or thousands of bands. If you were to visualize the genomic digest on a gel using ethidium bromide, the fragments would look like a smear of DNA because you will have fragments of every possible length. It would be impossible to isolate any one band with any degree of accuracy. Instead of trying to visualize these bands on a gel, the digested DNA is denatured into single-stranded DNA and transferred to a membrane. The probe is added, and it will anneal only to its specific complementary DNA sequence.
By running a DNA molecular weight marker, or "DNA ladder," you can approximate the size of band you are interested in. However, by designing a probe that spans the DdeI site in question, you can assure that your probe will anneal to two fragments in the wild-type allele (because the molecular probe has been cut in half at the same restriction site). If there is no DdeI site, you will have only one band. Thus, this strategy allows you to look at the total number of bands seen on the autoradiograph, not just their size differences.