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Figure 1. Strategy for mutation analysis. The structure of the human amelogenin gene is shown at the top. The exons are blocks numbered 1 through 7. Below each exon is a bar corresponding to the region amplified and a number indicating the number of nucleotides in each PCR product. The DNA sequences of the primer pairs used to generate the exon-specific PCR amplification products, written in the 5' to 3' orientation, are shown below, along with the annealing temperature used in the amplifications, provided in the table at the bottom. The PCR reactions have a five-minute denaturation at 94°C, followed by 40–50 cycles each with denaturation at 94°C for 30 sec, primer annealing at 56–61°C (as shown in the table) for 30 sec, and product extension at 72°C for 40 sec. In the final cycle, the 72°C extension was for 5 min. PCR amplification products were purified by use of the QIAquick PCR Purification Kit (Qiagen Inc., Valencia, CA, USA), and are shown on a 1% agarose gel stained with ethidium bromide, in the center of the Fig.
