T3/T7 PRIMER QUESTION!!!

Martin Kennedy mkennedy at chmeds.ac.nz
Tue Nov 8 21:25:38 EST 1994


In article <stauth-031194111951 at darenmac.usgmrl.ksu.edu>, stauth at crunch.usgmrl.ksu.edu (Darren Stauth) writes:
stuff deleted.......
> since T7 and T3 are so similar that T7 misannealed to the T3 site.  This
> leads me to my question:  since T7 and T3 are so similar and can misanneal
> to give amplification in PCR, will the same thing happen in sequencing
> reactions?  I've seen vectors containing both T7 and T3 sites.  Has anybody
> had problems sequencing in such a vector?
> Darren M. Stauth -- stauth at crunch.usgmrl.ksu.edu

I've never noticed mispriming, and I use T3/T7 a lot on Bluescript plasmids.  
I must admit, until you mentioned it I'd never noticed these things are 
quite similar, especially at their 3' ends:

    T3 primer  5'-ATTAACCCTCACTAAAG-3'
                       | ||||||| ||                      
T7 primer  5'-AATACGACTCACTCACTATAG-3'

With other primers I have noticed how a single mismatch will screw sequencing, 
but not affect PCR, so sequencing (at least with T7 polymerase) is more 
sensitive to subtle differences between template and primer than PCR.

Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
NN  NN NN           Christchurch School of Medicine            ZZZ
NN   NNNN              Christchurch, New Zealand              ZZZZZZZ



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