RAPD analysis in flax: Optimization of yield and reproducibility using klenTaq 1 DNA polymerase, chelex 100, and gel purification of genomic DNA
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We have developed an optimized RAPD analysis approach using the unusually heat-stable KlenTaq1 DNA polymerase. This enzyme is used in conjunction with a genomic DNA isolation method that includes a modified CTAB DNA isolation protocol, ethanol re-precipitation of resuspended nucleic acids from 2M NaCl, and Chelex 100 treatment. When needed, additional gel purification and isolation of high molecular weight DNA for use as a template in RAPD analysis is shown to remove amplification product ambiguity from within isolates of the same line as well as from between lines. This optimized RAPD analysis was used to define polymorphisms in lines of flax nearly isogenic for rust resistance at theL locus. It should also be useful for any plant species.
Key wordsRAPD KlenTaq 1 DNA polymerase CTAB DNA extraction Chelex 100 gel purification flax Linum usitatissimum
random amplified polymorphic DNA
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- Aldrich, J., and C.A. Cullis. 1992. 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. Plant Genome I, The International Conference on the Plant Genome, San Diego, CA. p. 16.Google Scholar
- Anonymous. 1991. Stoffel fragment: Increased thermal stability and broad Mg2+ optimum. Amplifications: A Forum for PCR Users, Issue 6: 14.Google Scholar
- Doyle, J. J., and J. L. Doyle. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bul. 19:11–15.Google Scholar
- Fang, G., S. Hammar, and R. Grumet. 1992. A quick and inexpensive method for removing polysaccharides from plant genomic DNA. Bio Techniques 13:52–54.Google Scholar
- Hosta, L., and P. Flick. 1992. Enhancement of specificity and yield in PCR. Comments, U.S. Biochemical Corp. 18:1–5.Google Scholar
- Singer-Sam, J., R.L. Tanguay, and A.D. Riggs. 1989. Use of Chelex to improve the PCR signal from a small number of cells. Amplifications: A Forum for PCR Users. Issue 3:11.Google Scholar