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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:

5'AATTGGATCC3'
    3'CCTAGGTTAA5'

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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2 Answers 2

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 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 GAATT`G` so it cannot recognize the site anymore. –  Chris Jan 30 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 at 7:23
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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
...NNNNNCTTAAGNNNNN...

          |
          V

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

          |
          V

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

          |
          V

...NNNNGaattggatccAATTCNNNNN...    after ligation
...NNNNCTTAAcctaggttaaGNNNNN...

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 at 17:06
    
Ok I have deleted. –  Alan Boyd Jan 29 at 17:14
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