Differential PCR based on 3' mismatch

Lars P. Ryder ryderlp at pip.dknet.dk
Fri Jun 20 02:47:05 EST 1997

Deborah Hailstones wrote:
>      Dear all,
>      We have been trying to use PCR to differentiate between two closely
>      related viroids, which differ from eachother by only a few
>      nucleotides.  We have designed PCR primers to these regions, placing
>      the differences at the 3' ends of the primers, to try to distinguish
>      between the viroids.  Sounds great on paper but the primers don't
>      actually seem to distinguish between them at annealing temp Tm - 5
>      oC.... 

What about trying: 

"Addition of a competitive primer can dramatically improve the
specificity of PCR amplification of specific alleles", BioTechniques
21:586-90 (1996).

Flemming Larsen 
Dept. of Clin. Immunol., sect. 7631
National University Hospital
Tagensvej 20
DK-2200 Copenhagen N

E-mail: flar at biobase.dk
phone:  +45 35 45 78 48
fax:    +45 31 39 87 66

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