Five dinucleotide microsatellites, D2S123, D5S107, D10S197, D11S904, and D13S175, were used as markers for the analysis of MSI and LOH.12 Using genomic DNA derived from tissue specimens, the five microsatellite sequences were amplified by polymerase chain reaction (PCR). Oligonucleotide primers corresponding to the microsatellite sequences12 were synthesized and purified by high-performance liquid chromatography. 5′ primers were labeled with the fluorescent compounds 6-carboxy-x-rhodamine or 6-carboxy-2′,4′,7′,4,7-hexachloro-fluorescein. PCR reactions were performed using Taq reagent kits (Takara Co, Ltd; Tokyo, Japan) and were run using a PCR system (GeneAmp PCR system 9600 or 2400; Perkin-Elmer; Norwalk, CT). Each 50 μL reaction mixture contained 1 times the reaction buffer, 350 μM each deoxynucleoside triphosphate, 10 pmol each primer, 2.5 U polymerase, and 25 μg genomic DNA. The thermal conditions of the system were as follows: one cycle at 95°C for 4 min; 35 cycles at 95°C for 0.5 min, 55°C for 0.5 min, and 72°C for 0.5 min; and 1 cycle at 72°C for 10 min. Then 0.5 U T4DNA polymerase was added to the mixture, followed by incubation at 37°C for 10 min. Each 1.5 μL product was mixed with 0.5 μL loading buffer (ie, blue dextran and 25 mM EDTA), 2.5 μL formamide, and 0.5 μL dH2O. To compare the electrophoretic profiles of two samples, 1.2 mL 6-carboxy-x-rhodamine-labeled product and 0.3 mL 6-carboxy-2′,4′,7′,4,7-hexachloro-fluorescein-labeled product were mixed. Samples were denatured and loaded onto a sequencer (model ABI 373A; Applied Biosystems; Foster City, CA). In each case, a size marker labeled with N, N, N′, N′-tetramethyl-6-carboxyrhodamine always underwent electrophoresis in each lane in order to standardize the mobility of the sample. The running conditions were 1,500 V, 20 mA, and 30 W for 5.5 h. The data were processed using computer software (ABI GeneScan; Applied Biosystems).