While using whole blood directly in PCR, we noticed strong inhibition by freshly collected samples (finger stick, 5-15 minutes after collection), compared to stored, frozen ones - at identical volume inputs. How to overcome this? This would be an important clinical aspect for point of care diagnostics, based on such technologies.
Hi, Iron containing proteins and heme groups are known to be PCR inhibitors.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC87763/
Iron binding proteins mainly contain Fe+2, which can bind oxygen. The oxygen is released in biological processes, which if not controlled, can denature proteins and kill pathogens. The Fe+2 in these proteins becomes Fe+3, which does not bind oxygen.
Frozen blood samples have less Fe+2. Maybe that is the difference.
Hi, I don't know if there is an oxidizing agent that specifically targets Fe+2 and does not oxidize other molecules.
I have seen in the literature that many use serum albumin to preserve a reducing environment. Bear in mind though, that serum albumin has one free -SH group. This free -SH of albumin is a major contributor to the reducing environment of plasma.
Many commercial preparations of albumin have oxidized -SH groups or the -SH as a dithiol with glutathione. Without a free -SH group, albumin is less effective at maintaining the redox of plasma.
AnyDirect buffer from BioQuest is successfully used with a variety of Taq polymerases specifically for blood substrates. There are several papers claiming it works great.
Yang YG1, Kim JY, Song YH, Kim DS.A novel buffer system, AnyDirect, can improve polymerase chain reaction from whole blood without DNA isolation.Clin Chim Acta. 2007 May 1;380(1-2):112-7.
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