Last year we reported a "modified dry blot" procedure (Simcox and McMullen, M, MNL 67:116-117, 1993), which is a modification of the standard Southern DNA transfer method (Southern, J. Mol. Biol. 98:503, 1975). The dry blot procedure simply involves using the buffer contained within the agarose gel to transfer DNA fragments onto a charged membrane. The entire process from removing the gel from the electrophoresis unit to pre-hybridizing the membrane requires less than 4 hours. Although many labs have been using a variation of the dry blot procedure to transfer plasmid DNA to membranes, the first report of the use of the dry blot technique in the analysis of complex genomes was just recently published (Kempter et al., TIG 7:109-110, 1991). The dry blot procedure is extremely simple, but effective.
Initially, the dry blot technique was used for F2 and interval mapping procedures in which membranes were stripped and re-probed numerous times. We have stripped several sets of membranes more than 18 times and re-probed with single copy RFLP probes, with no appreciable loss of signal. Membranes produced using the dry blot technique with single copy maize probes require a 1 -2 day exposure. The dry blot procedure has been used by different labs in a number of different applications with the same degree of success. In addition to RFLP analysis, we have used the dry blot procedure for CHEF analysis of YAC clones and high MW maize genomic DNA, co-segregation analysis using Mutator probes, and northern analysis.
Several changes were made to adapt the dry blot procedure for northern analysis. In one case, glyoxal-treated poly(A+)RNA from soybean somatic embryos was neutralized in 10 mM NaPO4, pH 6.5 and transferred onto Gene Screen Plus transfer membrane (Ma et al., Plant Mol. Biol. in press, 1994). Using this method, Hongchang Ma and co-workers detected expression of a soybean homeobox-containing gene, homologous to the maize knotted1 cDNA, after a 2 day exposure. Another variation involved equilibrating and transferring glyoxal-treated total RNA collected at different times after maize leaves were inoculated with Cochliobolus carbonum race 1, in 50 mM NaOH onto Hybond N+ transfer membrane (Gruis and Johal, unpublished). Several rare transcripts were detected using the Hm1 cDNA after a 3-4 day exposure.
We believe that the sensitivity of the dry blot procedure is derived from the use of charged transfer membranes. When other sources of transfer membranes are used, the method used to strip membranes after hybridization should be adapted according to the manufacturer's protocol. The procedure as described in the 1993 MNL article was developed using DuPont's Gene Screen Plus membrane. We found that stripping membranes with 0.4 M NaOH was far superior to high temperature treatments using high stringency solutions. (Names are necessary to report factually on available data; however, the USDA and the University of Missouri neither guarantees nor warrants the standard of the product, and the use of the name by USDA and the University of Missouri implies no approval of the product to the exclusion of others that may also be suitable.)
to the MNL 68 On-Line Index
Return to the Maize Newsletter Index
Return to the Maize Genome Database Page