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What would you do if you want to remove an EcoRI restriction site and introduce BamHI restriction site at apprx. the same location ?

One of the answers in my textbook was : To construct a DNA fragment with the structure:


I understand that this would ligate efficiently to the sticky ends generated by EcoRI cleavage and would introduce a BamHI site but I don't understand how this will remove/not regenerate the EcoRI site.

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up vote 3 down vote accepted

The recognition site for EcoRI is GAATTC, and the enzyme cuts after the first base. See this picture from NEB:

enter image description here

The overhang is: AATT, which is supplied by your oligo and fits into the overhang. If the next nucleotide whould be a C than the site would be recreated, since its a G its not. The new sequence of the old EcoRI site is GGATTG which is not recognized by EcoRI.

One technical note: To avoid religation of the digested DNA which carries this site, I would treat it with alkaline phosphatase to remove the phosphate end at the 5' end of the DNA.

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I am still not able to understand how it destroys the EcoRI site. Sorry for being so slow ! – biogirl Jan 30 '14 at 7:17
Look at the sequences: EcoRI needs GAATTC to recognize and cut DNA. After the ligation with your BamHI oligo the sequence of the site is GAATTG so it cannot recognize the site anymore. – Chris Jan 30 '14 at 7:19
I get it now. I also understood why I couldn't figure it out at first - because I thought the RE just cares about G and A not any further sequence but now I understand that the whole sequence is important. THANKS A TON ! – biogirl Jan 30 '14 at 7:23

Just in case you are having difficulty visualising what happens in the answer from @Chris, here are the steps with the linker shown in lower case

...NNNNNGAATTCNNNNN...           ds DNA


...NNNNG          AATTCNNNNN...  after EcoRI digestion
...NNNNCTTAA          GNNNNN...


...NNNNG      aattggatcc        AATTCNNNNN...   incoming linker
...NNNNCTTAA      cctaggttaa        GNNNNN...    


...NNNNGaattggatccAATTCNNNNN...    after ligation

The left hand "EcoRI" is now GAATTG and the right hand "EcoRI" is CAATTC.

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I was wrong reading the sequence. Removed that part. – Chris Jan 29 '14 at 17:06
Ok I have deleted. – Alan Boyd Jan 29 '14 at 17:14

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