Free energy of hairpins and their Tm

Tue Jun 18 06:21:30 EST 1996

Dear readers,

I have a problem in finding information on the relation between the free 
energy of hairpins and the conditions under which they do not form.
As for an example, I am screening a cDNA library to find a pig homologue to 
the human angiotensinogen gen. Though I have obtained three independent 
clones of another gene, known to be low abundant in that tissue, screening 
500,000 plaques, I still did not find any clone of angiotensinogen after 
screening 2 million plaques. In the human sequence there is a hairpin in the 
3' untranslated region about 300bp upstream of the polyadenylation signal. 
The free energy is -7.8 Kcal/mol. Now I am wondering if the reverse 
transcriptase would have been able to read 'through' this hairpin i.e. at 
what temperature will this hairpin not form.
Another example concerns PCR. In my constructs I find hairpins with free 
energies in the range from +4.7 down to -12.2 Kcal/mol. Is there a rule to 
determine what Tm must be used to circumvent a hairpin with a specific free 
energy in PCR?
I hope someone can give me/us some clues on this topic.
Thanks in advance!
Best wishes,

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