Figure 2. Identity of PDEA-L clone as PDEa cDNA sequence

(A) Relative size of the inserts in the PDEA-L and PDEA-S clones, the DNA fragments amplified by PCR, primers (see Table 1 for sequence) used, and the predicted sizes of the amplified fragments are shown. PCR condition for amplified fragments are as follows: fragments 1 and 2 were obtained by 30 cycles at 94 °C (1 min), 64 °C (1 min), 72 °C (2 min); fragment 3 was obtained by 30 cycles at 94 °C (1 min), 54 °C (1 min), 72 °C (1.5 min); and fragment 4 was obtained by 35 cycles at 94 °C (1 min), 58 °C (2 min), 72 °C (3.5 min). All reactions were concluded with a single step extension reaction at 72 °C for 5 min.

(B) Agarose gel (0.8%) electrophoresis of PCR products from PDEA-S (S) and PDEA-L (L) clones. Numbers 1 through 4 identify DNA fragments amplified from regions of the clones represented in panel A. Lane M1 represents BstE II-digested lambda DNA markers

(C) Polyacrylamide gel (6%) electrophoresis of restriction enzyme digestion of PCR product (4 in panel A) from PDEA-S (S) and PDEA-L (L) clones. Lane M2 represents Hae III-digested phi X 174 markers. The length of the DNA fragments (bp) in both markers (M1 and M2) are shown next to the marker lanes. The PCR and the restriction enzyme digestion results indicate that both the clones are identical in the regions examined.