However, what sets
our protocol apart from automated or radioactive ones is the use
of biotin. Biotin is a compound that we incorporate into the primer,
(used to elongate replicating strands of DNA), which can be detected
through a streptavidin-alkaline-phosphatase wash. The biotin binds
to the DNA fragments while the streptavidin alkaline-phosphatase
wash latches onto the biotin. Using X-ray film, the DNA fragments
can be captured because the wash cleaves phosphates that emit light.
This particular method is called chemiluminescent detection.
A hurdle we have faced with
this protocol is keeping the biotin from degrading at the high temperatures
necessary to denature the DNA so that biotin-labeled primers can
attach to the fragments. We've slightly altered the protocol so
that the biotin survives denaturation.