International Mammalian Genome Society

The 16th International Mouse Genome Conference (2002)


POSTER 164 - AflII FRAGMENTS ARE HIGHLY VARIABLE AMONG MOUSE STRAINS: A NEW APPROACH USING TWO-DIMENSIONAL DNA ELECTROPHORESIS

J Asakawa
Radiation Effects Research Foundation

2) Shiroishi T, 1) Nakamura N
1) Radiation Effects Research Foundation, 2) National Institute of Genetics

We found that a two-dimensional DNA electrophoresis (2-DE) method permits visualization of numerous DNA fragments highly variable among mouse strains.  In this method, we used AflII restriction enzyme (C/TTAAG) as the landmark enzyme in place of ordinary NotI.  Genomic DNAs were first digested with AflII/HaeIII (GG/CC), 3-8kb fragments were isolated and radio-labeled at the AflII sites with a-32PdTTP and a-32PdATP.  After the first dimension electrophoresis, the DNA fragments were further digested in situ with HinfI and separated perpendicularly by the second polyacrylamide gel electrophoresis.  Gels were then dried and autoradiographed.  Approximately, 1100-1200 fragments (spots) were visualized on a single gel from inbred strains.  Analyses of 2-DE patterns showed about 10% strain-specific AflII spots between genetically closely-related strains, DBA1 vs. DBA2 or CBA vs. C3H.  The fraction of strain-specific spots increased over 30% between strains of laboratory mice, e.g., C3H and C57BL/6, and reached to over 80% between BALB/c and JF1, an inbred strain originated from the Japanese wild mouse, M. m. molossinus.  The frequency of inter-strain variations seemed to be comparable to that of microsatellite, while our preliminary study indicate that the present AflII fragments do not comprise simple repeat sequences.


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