Abstract
This chapter describes a method for the construction of a cDNA library with recently developed refinements in addition to the structure and evolution of calcium protease subunits revealed by the eDNA cloning. Calcium-dependent protease is an intracellular nonlysosomal protease that requires calcium for its proteolytic activity. Two types of calcium protease, calcium protease I and II, respectively, requiring micro- and millimolar orders of Ca2+, exist in mammalian tissues. There have been important refinements in several stages of cDNA cloning procedure as the conventional method has been established. These include the combined use of DNA polymerase I and RNase H in the second-strand cDNA synthesis and the use of λ phage-based cloning vectors. For these reasons, it has become easier to isolate full-length eDNA clone for rare and long mRNA species, if one has a good probe for the detection. The strategy involves the construction of large numbers of cDNA clones from total poly(A)+ mRNA preparations and the identification of the cDNA clone of interest.
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