Linear Restriction map PDF

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Linear Restriction map...

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Linear Restriction map The following is a restriction mapping question involving linear DNA with a triple digest. There is a circular restriction digest power point on Angel as well. Using the resulting fragment sizes, we can draw what the linear restriction map including all three enzymes looks like. For this exercise, it will be helpful to remember that 1 KB = 1000 bp To begin, it is easiest to pick a restriction enzyme that cuts the most times. That would mean starting with BamI. A linear 18 kb fragment of DNA is digested with three restriction enzymes: HindIII, BamHI, and EcoRI separately and in each possible pair. You run out the results of each digestion on an agarose gel and see bands of the following sizes. How many times does each enzyme cut the linear DNA? Draw the restriction map that illustrates all three enzymes. HindIII: 100 bp, 1700 bp BamHI: 300 bp, 400bp, 1100bp EcoRI : 900 bp HindIII + BamHI : 100 bp, 300 bp, 1100 bp HindIII + EcoRI: 100 bp, 800 bp, 900 bp BamHI + EcoRI: 300 bp, 400 bp, 500 bp, 600 bp

We know that BAMI results in fragments of sizes: 300 bp, 400bp, 1100bp. So we draw that on a linear map of DNA that is in total 1.8 kb. Since there are a total of 3 fragments, we know that this enzyme cuts the DNA twice, as shown above. If we plot cut sites at 300 bp & 1400 bp, that will give us 3 bands of the desired lengths. These fragments are illustrated above in red. From left to right: The first band is from the first cut at 300 bp to the beginning, or 300 – 0 = 300 bp. The second band is from the first cut at 300 bp to the second cut at 1400 bp, or 1400 - 300 = 1100 bp. The third band is from the last cut at 1400 bp to the end, or 1800 – 1400= 400 bp. Check work: 300 + 1100 + 400 = 1800.

Now let’s do HindIII. We know that HindIII results in fragments of sizes: 100 bp and 1700bp. So we draw that on a linear map of DNA that is in total 1.8 kb. Since there are a total of 2 fragments, we know that this enzyme cuts the DNA once, as shown above. If we plot a cut site at 1700 bp, that will give us 2 bands of the desired lengths. These fragments are illustrated above in red. From left to right: The first band is from the only cut at 1700 bp to the beginning, or 1700 – 0 = 1700 bp. The second band is from the only cut at 1700 bp to the end, or 1800 – 1700= 100 bp. Check work: 1700+ 100 = 1800

Now let’s do the 3rd enzyme, EcoRI. We know that EcoRI results in one fragments of size: 900 bp. This one is a little tricky. There is only one band, which at first leads us to believe that the DNA has not been cut at all by this enzyme. However, if that were the case, we would see one band of 1800 bp, not one band of 900 bp. What is going on here is that the enzyme has cut the DNA exactly in half, giving us two fragments of the same size. We cannot visualize this on a gel, since fragments of the same size

will be on top of each other, and there’s no way to determine if there is more than one band by visualization alone. However, by looking at the sizes we are giving, we can think this through. If we plot a cut site at 900, that will give us 1 band (but 2 actual fragments) of the desired length. These fragments are illustrated above in red. From left to right: The first band is from the only cut at 900 bp to the beginning, or 900 – 0 = 900 bp. The second band is from the only cut at 900 bp to the end, or 1800 – 900 = 900 bp. Check work: 2 x 900 = 1800.

Now we want to put these all together. Let’s start with the first combination: HindIII + BamI, which we know gives us fragments of sizes: 100 bp, 300 bp, and 1100 bp.

If we plot all of these on the same piece of DNA, it looks like the above. We get 4 fragments: 1 fragment of 100 bp, 2 of 300 bp, and 1 of 1100 bp.

Now if we add EcoRI to the previous map, it looks like the above. We get 5 fragments: 1 fragment of 100 bp, 2 of 300 bp, 1 of 500 bp, and 600 bp. Now let’s check this against the information we were given in the above.

Check all possible combinations to make sure that the math is correct, and you have fragments of the appropriate sizes....