Plant Genome I Conference
Town & Country Conference Center, San Diego, CA, November, 1992.
PG-I: 29pg1
ENHANCED SCREENING OF RAPDS IN FLAX LINES NEARLY ISOGENIC FOR
THE L RESISTANCE GENE USING LOW MELTING AGAROSE FOR DNA
PURIFICATION AND KLENTAQ 1 FOR PCR
Jane Aldrich 1 and Chris Cullis 2 1Lakeside Biotechnology, Inc.
Chicago, IL 60612; 2 Biology Dept., Case Western Reserve
University, Cleveland, OH 44106
Differential (D) lines of flax containing different alleles
at the L locus were constructed by Flor in 1956 by back-crossing
source lines to Bison 8 - 12 times. These lines are being
compared to Bison for detection of RAPDs using random 10-mers as
primers. DNA isolation from these lines is complicated by the
presence of polysaccharides and secondary metabolites that
interfere with PCR. DNAs were isolated from older plants as well
as from 3-week old seedlings using a CTAB DNA isolation
procedure. In contrast to DNA isolated from older plants, all
DNAs isolated from seedlings were readily cleaved by EcoRl and
HindIII, indicating that polysaccharide contamination was
minimal. However, DNA of the same D line isolated from seedlings
grown at different times produced variability in RAPD pattern
with some primers. We found that the only approach that removed
the variability in band production within a line was isolation of
high molecular wt. DNA from low melting agarose. Then all D
lines and Bison had the same banding pattern. We have also
compared the truncated Taq DNA polymerases Stoffel fragment,
KienTaq 1 and KlenTaq 5, and have found KienTaq 1 to be superior
in the quality and reproducibility of PCR products formed.
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