TY - JOUR
T1 - Precise assessment of microsatellite instability using high resolution fluorescent microsatellite analysis
AU - Oda, Shinya
AU - Oki, Eiji
AU - Maehara, Yoshihiko
AU - Sugimachi, Keizo
N1 - Funding Information:
We thank K.Takahashi, M.Sakabe and Y.Ohkuma (Perkin Elmer Japan Co., Ltd Applied Biosystems Division) for technical support. We are most grateful to M.Sekiguchi for helpful advice and to M.Ohara for comments on the manuscript. This study was supported by grants from the Ministry of Education, Science, Sports and Culture of Japan.
PY - 1997/9/1
Y1 - 1997/9/1
N2 - 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.
AB - 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.
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U2 - 10.1093/nar/25.17.3415
DO - 10.1093/nar/25.17.3415
M3 - Article
C2 - 9254697
AN - SCOPUS:0030851907
VL - 25
SP - 3415
EP - 3420
JO - Nucleic Acids Research
JF - Nucleic Acids Research
SN - 0305-1048
IS - 17
ER -