A protocol from a Paper says they extend PCR at 60 degrees. Is this possible?
Difficult to say, in the handbook http://www.qiagen.com/literature/render.aspx?id=367 (short version: http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&cad=rja&ved=0CFQQFjAC&url=http%3A%2F%2Fwww.qiagen.com%2Fliterature%2Frender.aspx%3Fid%3D201425&ei=fq61UMbqHMem4ATcooCACg&usg=AFQjCNHvkpc7SF6zizsSuwaYxstrnL_nPQ ) there is the information that the extension works at 68°C. But PCR is every time a little bit of vodoo ;-) Just try it (in my lab we use 68°C)...
qiagen long range PCR enzyme works best at 68oC..but theoretically it is very possible that it will work at 60oC ..Only issue is that Tm should be low enough not to overlap with extension temp and there are chances of non specific amplification which can be avoided by adding 5% DMSO.. one more thing at low temp , extension time must also be increased to accomplish amplification..
Please do not mix extension temperature and Tm. Tm is the temperature where the primer still attached to the template DNA. As higher as better because lower chance for non-specific amplification. Extension or elongation temperature is the temperature where the polymerase still can work. They are thermostabil (they have to be!) but it does not mean that they are not lost activity at high degree. A normal PCR use 72°C and 15-30 seconds of elongation time, but in case of long fragment amplification also the elongation time is longer, consequently they will lost activity faster than in case of short fragment. Hence they are using only 68°C to save the enzyme. Usually in this case also the denaturation degree is lower!
There is no other objection against 60°C elongation temperature as that the non-specific amplification could be higher. But not necessarily. If the primer has a high enough Tm temperature and high binding at the 5`part, it should work. Even you can use a two step PCR where the annealing and elongation happens at the same temperature.
Agree with Maria Berenyi.
If for any reason you need to use 60C - do it - it is called two step PCR (95- 60).
But I am suspecting that you missed the point while read the manual; 60 C is for annealing of primers and then you proceed to 68C when the extension is happening.
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