Rules for insertion of EBV OriP into expression vector?

Alan KAY kay at lyon151.inserm.fr
Thu Jan 9 10:43:33 EST 1997


In article <slrn5d7lkd.j13.aiyar at ebv.oncology.wisc.edu>, 
aiyar at ebv.oncology.wisc.edu says...
>
>On 8 Jan 1997 16:07:05 GMT, Alan KAY <kay at lyon151.inserm.fr> wrote:
>
>>Without knowing if there are any rules, I would tend to think that having 
>>active transcription through an origin of replication may not be too good a 
>>thing, with transcription affecting the DNA double helix at that place. 
>
>This is probably true, although papers from Bruce Alberts and his colleagues
>would lead one to believe that it is not a problems.  In the case of oriP,
>one should keep in mind that since it (oriP) fires only *once* per cell 
>cycle, the probability of a collision between transcription and replication 
>forks is not very high.
>
>>I think the best way would be to amplify the EBV ori by PCR using 
>>primers that also include a restriction site(s) that you can use to clone 
the 
>>fragment somewhere else in the plasmid where it is less likely to do harm.
>
>This is likely to be difficult.  We have been unable to amplify oriP
>by PCR.  OriP has twenty copies of a 20 nt repeated element, called
>the family of repeats, which in our hands is recalcitrant to PCR
>amplification.
>
>Ashok
>-- 
>Ashok Aiyar, Ph.D.
>McArdle Laboratory for Cancer Research
>University of Wisconsin-Madison


In this case, if PCR is not possible, then Kevin should excise oriP, blunt 
end the excised fragment and clone it into a suitable restriction site in the 
receptor vector which has also been blunt ended. Alternatively, if there are 
no suitable restriction sites in the region of the vector where he wants to 
put oriP, then he should create a unique site, preferably either compatible 
with the ends of the excised oriP fragment or which is blunt ended.

Alan KAY




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