Figure 3.

Flanking PCR is limited to detecting molar ratios of 1:10 to 1:100 of alternative to constitutive templates. Abbreviations: C and A lanes represent constitutive and alternative template controls. A titration was done of different ratios of the alternative and constitutive splice forms beginning with a one-to-one molar ratio of 50 000 copies each, followed by serial dilutions of 1:100,1:1000 and 1:10 000 of alternative to constitutive templates. 40 cycles of PCR were used for both experiments. (A) Most of the alternative exons could not be detected beyond a 1:10 dilution of alternative to constitutive when using flanking PCR. Here, only one case showed that the flanking PCR was able to detect the alternative in at most a 1:100 molar ratio (bottom gel). (B) The exon-specific PCR approach demonstrated the greatest sensitivity in all three cases, detecting the alternative isoform even when the constitutive form was present at a 10 000-fold excess. DNA ladder λ shows 50 bp, 75 bp, 100 bp, then 50 bp increments to 350 bp, then 500 bp and 766 bp. The strongest band represents 200 bp.