Representative autoradiographs of microsatellite analysis (A) and standard curves for methylation and unmethylation MSP amplification (B) using a radioisotope. (A) A "multiplex, hot-start" method was applied to the PCR-based loss of heterozygosity (LOH) analysis. Forty microsatellite allelic loci were amplified in 25 reaction mixtures each of which contained one (10 mixtures) or two (15 mixtures) pairs of primers. A mixture of different-sized two to four amplicons was loaded onto one lane. A total of 80 microsatellite amplicons from each specimen were run simultaneously on one sequencing gel. Case 1 had high-level LOHs involving chromosome 4p, 5q, 9p, 13q, 17p, and 18q. The normal (N) and the corresponding tumor (T) DNAs are indicated above or below each microsatellite amplicon. The asterisk indicates a LOH. (B) The genomic DNA universally methylated by DNA methylase (CpGenome Universal Methylated DNA, Chemicon, Temecula, CA) was used as the methylated control DNA. The PCR DNA, which had been amplified by the universal primer (5'-CCG ACT CGA GNN NNN NAT GTG G-3'), was used as the unmethylated control DNA. Variable mixtures of the two opposite control DNAs according to their PCR intensity of 20 ng/μl were amplified using a set of MSP primers for the non-island CpGs of the TFF2 gene. The proportion of methylated and unmethylated CpGs was calculated using the following formula: Methylation or unmethylation proportion (%) = (methylation or unmethylation intensity/(methylation + unmethylation intensity)) × 100. The relative band intensities of the methylation (M, closed circle) and unmethylation (U, open circle) primer set were plotted as a function of the control DNA content. The amplification intensity ratio of the MSP primer set increased linearly with increasing percentage of the corresponding control DNA in the MSP mixtures.