Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.
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You say "The intron has a mutation which has caused it to increase in length (by several kb)." If the intron, as opposed to one of its restriction fragments, is larger due to a mutation, that implies an insertion mutation. If one or more restriction fragments have increased in size, that could be still an insertion, or it could be point mutations.
If you only see a restriction fragment has "shifted" upward on the gel, then there is likely an insertion in that fragment of the intron. The inserted DNA does not contain a restriction site for that particular enzyme, and the insertion is causing the fragment to be larger by the size of he inserted DNA. If the insertion did contain one restriction site for the given enzyme, then you would likely see two new fragments (bands on the gel.) You would probably see one fragment that was in your original DNA disappear, i. e., the fragment that contains the insertion in the mutant. If the insertion contained more than one restriction site within it, or created a new site at its insertion point, it gets real messy, real fast.
If when you say "increase in length," you are referring to the size of a restriction fragment, not actually to the intron itself, then we are more likely talking something different. If a new restriction fragment appears when the mutation is present, and you see one or two smaller fragments disappear, the mutation was likely a point mutation (single base change) at the restriction site. The size of the fragments that disappear should add to the approximate size of the new larger fragment. Conversely, if the point mutation caused (created) a restriction site, then you would see a larger fragment disappear, and one or two smaller fragments appear. Again, the size of the larger fragment should approximate the sum of the sizes of the smaller fragments.
There are other possible scenarios, such as deletions, but those I described seem the most likely and will hopefully give you some ideas as to what to look for. Remember, restriction mapping is only looking at the approximate sizes of various fragments of DNA. Two different fragments can be the same approximate size, and you may only see one band. In the perfect world, that band will appear more intense, but not always.
Restriction mapping is a comparative analysis. You should be comparing the unmutated intron's fragment lengths to those of the mutated intron's fragment lengths. You would be comparing the restriction map of the known (unmutated) with the sizes you get from running the restrictions of the unknown (mutated.) If this was really being done in a lab, you would have absolute sizes of fragments (bp or kb) as determined by comparison to a size standard. The sizes are approximate, but absolute, i.e., not relative. Since you are saying the intron gets larger, that implies an insertion. The restriction map should give you the approximate size and location of the insertion. If you do not know the map or fragment lengths of the unmutated, there is not much you can say about the mutated. You could create a map of the mutated intron, but that info is not particularly useful without something to compare it to.
4 posts • Page 1 of 1
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