Hello Every one, I am wondering whether southern blot can determine the mutated genes while they will be used as probes. Will it show result if the DNA fragment used as Probe has mismatches at intervals. Secondly, If the probe is 1KB long, but 700 bp is same, but 300 bp are not same on either of the sides. Will southern blot give the result or not? If yes, what is the possible solution to get a accurate band. Thank you.
Southern blotting is more often used for tracking linked markers through families to indicate which unknown members have, or have not got a mutation but the probe only needs to be close to the gene of interest. 700 bases is a long probe so will anneal well unless the washing steps are very stringent, If the mutant gene is linked to an RFLP then Southerns are very useful.. Mismatches in the probe simply mean that wash steps should be low stringency .. You can often determine the wash conditions by dot blotting your dna on to nylon membranes and washing under different stringencies like 6x SSC to 0.2 x SSC before doing the gel separation of digested dna and blotting on to the membrane which is labour intensive but will have to be done if you are tracking genes or markers through families
I am not sure if I understood the application your are planning for your Southern blot experiment...
For example, Southern blot can be used to find gene orthologs in other species (zoo blot), by using carefully selected conditions for hybridisation and washes.
By appropriately choosing the restriction enzymes, the labelling method for the probe and hybridisation conditions, you may be able to distinguish small duplication/deletions. If you want to determine the presence of point mutations, however, a dot blot and oligo labelling may be more appropriate, as with short oligos (20-25 nt) a mismatch will more easily destabilise hybridisation. Alternative methods (PCR ARMS etc) can be used as well, depending on how many mutations you are searching.
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