Plant Genome I Conference
Town & Country Conference Center, San Diego, CA, November, 1992.
PG-I: APPLYING 4-COLOR FLUORESCENT TECHNOLOGY To DNA FINGERPRINT
ANALYSIS OF PLANT GENOMES
APPLYING 4-COLOR FLUORESCENT TECHNOLOGY To DNA FINGERPRINT
ANALYSIS OF PLANT GENOMES
J. S. Ziegle and S. Koepf, Applied Biosystems, Inc. Foster City,
CA.
Researchers in several laboratories have shown random
amplification with arbitrary primers to be especially useful with
plant genomes where not much sequence information is known. The
amplification of a given genome with a single primer will result
in a reproducible number of DNA amplification products of various
sizes. These amplification products are then size separated by
electrophoresis and the resulting pattern of bands can then be
visually compared to other strains, species, etc. Martin, et.
al. (1991) have used single primer amplification to rapidly
isolate DNA sequences that are linked to resistance genes in
tomato. By comparing the DNA fingerprint profiles of
near-isogenic lines (NIL), polymorphisms can be detected. NIL
differ only by the presence or absence of a target gene and a
small region of closely linked DNA. Therefore any polymorphisms
detected should be linked to that region. The study describe in
this report was designed to compare 4 NIL of rice, three of which
are resistant to Xanthamonas oryzae pv. oryzae, which causes
bacterial blight of rice. Genomic DNA of these 4 lines were
amplified with 20 separate 10-mers in an effort to identify
polymorphisms. Four-color fluorescent technology has been
applied to the analysis of PCR amplification products of
polymorphic microsatellite loci. In this report we describe the
application of this technology to the analysis of DNA
fingerprints. PCR products were dye-labeled during amplification
with a single 5'- dye labeled oligomer. The dye-labeled
fragments are elegtrophoresed past a scanning laser beam and the
emitted fluorescence is detected in real time. Software
automatically determines the size of the PCR products from each
amplification by comparison to an internal lane size standard.
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