PCR - reduced binding efficiency with lower Tm? - (Feb/29/2008 )
I know that the usual rule for PCR is that a higher melting temperature increases the specificity of primer binding (and makes it more difficult for primers to bind to a less-than-perfectly-matching sequence) and vice versa (so at a lower temp you may get multiple bands from lot of unspecific binding).
Has anyone ever heard of the opposite happening?? I've been having trouble with a certain primer set for a while and in some cases, a lower melting temp results in negatives that were positives at a higher melting temp. I'm very confused. 
-Paralith-
QUOTE (Paralith @ Feb 29 2008, 01:43 PM)
I know that the usual rule for PCR is that a higher melting temperature increases the specificity of primer binding (and makes it more difficult for primers to bind to a less-than-perfectly-matching sequence) and vice versa (so at a lower temp you may get multiple bands from lot of unspecific binding).
Has anyone ever heard of the opposite happening?? I've been having trouble with a certain primer set for a while and in some cases, a lower melting temp results in negatives that were positives at a higher melting temp. I'm very confused.
Has anyone ever heard of the opposite happening?? I've been having trouble with a certain primer set for a while and in some cases, a lower melting temp results in negatives that were positives at a higher melting temp. I'm very confused.
Are you sure if what you see as positive is really positive? It' couldn't happened that what you see is inespecif and when you increase the temperature dissapear??
-marielita-
QUOTE (Paralith @ Feb 29 2008, 08:43 AM)
I know that the usual rule for PCR is that a higher melting temperature increases the specificity of primer binding (and makes it more difficult for primers to bind to a less-than-perfectly-matching sequence) and vice versa (so at a lower temp you may get multiple bands from lot of unspecific binding).
Has anyone ever heard of the opposite happening?? I've been having trouble with a certain primer set for a while and in some cases, a lower melting temp results in negatives that were positives at a higher melting temp. I'm very confused.
Has anyone ever heard of the opposite happening?? I've been having trouble with a certain primer set for a while and in some cases, a lower melting temp results in negatives that were positives at a higher melting temp. I'm very confused.
In suppression-PCR (only a single primer corresponding to the inverted terminal repeats of the target was used), decreased annealing temperature may reduced the efficiency of primer-template annealing.
The two papers listed below may help.
1.Regulation of average length of complex PCR product.
http://nar.oxfordjournals.org/cgi/content/full/27/18/e23
2. PCR-suppression effect: kinetic analysis and application to representative or long-molecule biased PCR-based amplification of complex samples.
http://www.sciencedirect.com/science?_ob=A...0885ddef96d2856
-zhongmindai-