Figure 3. HbIII cDNA Primer Extension Analysis. (A). Schematic Diagram of Primers Design.

Two oligonucleotide primers complementary to a sequence within 72 and 101 nt downstream of the anticipated 5′ end of mRNA sequence were end labeled and used in primer extension reactions as described in section 2.6. (B) Primer Extension Analysis. 0.5-0.6×10⁶cpm of radiolabeled primer was added to 15 μg of total RNA. Primer extension was done using the rTth Reverse Transcriptase. The radiolabeled cDNA was analyzed through an 8% denaturing polyacrylamide gel and co-electrophoresed with pBR322cut with MspI. Lane 1: ³²P end-labeled pBR322 vector cut with MspI, Lane 2: Primer Extension using HbIIIPE1 and Lane 3: Primer Extension using HbIIIPE2. The sizes of fragments in marker and sample lanes are expressed in nucleotides (nt).