G or C at 3' end of primer?

Michael Szardenings msz at bmc.uu.se
Tue Jun 4 08:40:25 EST 1996


In article <4oet96$ch5 at sunca.ncaur.gov>, skorycd at ncaur1.ncaur.gov says...
>
>I have always made my PCR and sequencing primers with a G or C 
>at the 3' end to presumably stabilize it during extension.  While I am 
>not aware of any research papers to support this logic, I have seen 
>several manufactures (eg: PerkinElmer) recommend a similar 
>design.  
>

Dear Chris,
it makes sense, but in most cases you won't notice the difference. If your 
gene is very rich in GC's a 3'end like x(N)ggCCg for example may lead to 
unspecific priming just with this end. The reason to avoid stretches is the 
fact, that there is some bias towards long stretches of 5 or more 
identical nucleotides. This is worst for G/C, besides the A/T's resulting from 
polyadenylation sites in cDNA libraries. There was some paper about this many 
years ago in JMB(?). As the polymerase will remove the first 3' base when 
starting polymerisation, it may not be enough to add only one base beyond such 
stretches in your primer to maintain specifity.
5' does certainly not matter, but your oligo should be stable still. I found 
Oligo4.0 very helpful in predicting the TM, especially to avoid too weak or 
too stable primers (TM>>60 deg C). The secondary structure options of these 
programmes are certainly helpful, but I agree with the all the other RE:, that 
by eye inspection very often is enough.
G.L.		Michael
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