The complete structure gene of the human 2ar of subjects with known phenotype (2ar density) was amplified using the established PCR protocols (see section PCR), and cloned into a pUC18 (see section puc and figure fig:puc) vector. As the transferred insert had a length of 1397 base pairs, it was not practicable to sequence it in one run. Although it would have been possible to sequence the insert using commercially available Forward and Reverse primers only, a smarter solution to the problem was to subdivide the coding region in easily reproducible overlapping 200 to 400 bp long sequence sections. This made it possible to read found mutations several times. The primers used and the starting points for the sequence reaction are summarized in table 3.1.
Table: Sequencing primers for the 2ar