International Mammalian Genome Society

The 15th International Mouse Genome Conference (2001)


POSTER 215 - USE OF INTER-SIMPLE SEQUENCE REPEAT PCR ANALYSIS IN MOUSE CARCINOGENESIS STUDIES

Dr Fernando Benavides
Department of Carcinogenesis
The University of Texas
M.D. Anderson Cancer Center
Science Park-Research Division
Smithville, TX 78957 U.S.A

Co-Authors: Zamisch M, Flores M, Escandon J, Sternik G, Richie E, Conti C
Institution: Department of Carcinogenesis, The University of Texas M.D. Anderson Cancer Center

Genomic instability is believed to play a significant role in cancer development by facilitating tumor progression and tumor heterogeneity. Inter-simple sequence repeat (Inter-SSR) PCR has been proven to be a fast and reproducible technique for quantitation of genomic instability (amplifications, deletions, translocations and insertions) in human sporadic tumors. The genomic instability detected by this technique is independent of the replication error phenomenon (microsatellite instability) because only inter-microsatellite DNA segments are analyzed. Inter-SSR PCR has been adapted in our laboratory for the analysis of chemically induced mouse tumors using (CA)8RY, (CA)8RG, (AAC)6Y and (AGC)4Y primers. We established the best PCR conditions for each primer and critically evaluated the reproducibility of the band patterns and the absence of spurious bands. We studied the stability of the fingerprints within mouse inbred strains and decided to use normal DNA from the same animal harboring the tumors. Even though inbred strains and hybrid F1 mice are, by definition, isogenic, we have found some level of intensity changes in electrophoretic bands between individuals. PCR was carried out as follows: initial denaturation 3 min at 94C, 30 cycles of 30 sec at 94C/ 45 sec at 54-58C/ 2 min at 72C, final extension 7 min at 72C. PCR products were analyzed by electrophoresis through 8 or 10% polyacrylamide gels and silver staining. Each individual primer generates a unique set of approximately 20 PCR products (less than 1 kilobase in size).

Tumor specific alterations were detected as gains, losses or intensity changes in bands when compared with matched normal DNA. We quantitated the extent of alterations by dividing the number of altered bands in the tumor by the total number of bands in normal DNA, creating an "instability index". Examples of genomic alterations analyzed by inter-SSR PCR of N-methyl-N-nitrosourea (MNU)-induced thymic lymphomas are presented. Instability index values ranged between 0-70%, the highest levels observed in MNU-induced thymic lymphomas generated in p53 nullizygote (-/-) mice. These PCR-based multilocus fingerprints provide a novel approach to assess the number of mutational events during tumor development in spontaneous and induced mouse tumors.


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