Precise assessment of microsatellite instability using high resolution fluorescent microsatellite analysis.
The instability of microsatellite sequences dispersed in the genome has been linked to a deficiency in cellular mismatch repair. This phenotype has been frequently observed in various human neoplasms and is regarded as a major factor in tumorigenesis. To demonstrate alterations in microsatellite sequences, polymerase chain reaction (PCR) and electrophoretic analysis are techniques often used. However, the electrophoretic profiles of PCR-amplified microsatellite sequences have not been well characterized. Moreover, the conventional method using autoradiography has critical problems in detection characteristics and migration accuracy. We made use of fluorescence-labeled PCR and laser scanning with linear detection characteristics, so as to detect bands quantitatively. Next, we characterized Taq polymerase-dependent modification of the amplified microsatellite sequences, using artificially synthesized microsatellite alleles and we optimized the electrophoretic profiles by enzymatic modification with T4 DNA polymerase. We developed a dual fluorescence co-electrophoresis system, in which both samples derived from cancer and normal tissues are electrophoresed in the same lane, in order to minimize migration errors. These improvements remarkably facilitate precise and objective assessments of microsatellite instability. Analyzing many positive cases in cell lines and tissue specimens, we classified all the patterns of microsatellite alteration and set up new criteria for assessing microsatellite instability.
Oda
S
S
Cancer Center, Kyushu University Hospital, Fukuoka 812-82, Japan. shinya@cancer.med.kyushu-u.ac.jp
Oki
E
E
Maehara
Y
Y
Sugimachi
K
K
D000230:Adenocarcinoma / Q000235:genetics; D003110:Colonic Neoplasms / Q000235:genetics; D004247:DNA / Q000032:analysis / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase; D004355:Drug Stability; D005456:Fluorescent Dyes; D006801:Humans; D018895:Microsatellite Repeats; D009369:Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction; D019914:Taq Polymerase; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000378:metabolism
D005456:Fluorescent Dyes; D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase