|Richard Jr, Edward|
Submitted to: American Society of Sugar Cane Technologists
Publication Type: Abstract Only
Publication Acceptance Date: May 17, 2004
Publication Date: June 16, 2004
Citation: Pan, Y., Veremis, J.C., Scheffler, B.E., Tew, T.L., Grisham, M.P., White, W.H., Richard Jr, E.P. 2004. High-throughput application of the DNA marker technology in sugarcane breeding: Phase I - DNA extraction [abstract]. Journal of the American Society of Sugar Cane Technologists. 24:117. Technical Abstract: Nucleic acid extraction is the primary limiting step in high-throughput (HT) application of the DNA marker technology in modern sugarcane breeding. A common method of extraction is to homogenize the sugarcane leaf tissue in a CTAB buffer with a Mini-Beadbeater followed by organic solvent fractionation and alcohol precipitation. Using this method, a technician can process approximately 60 DNA samples per day. Although the quantity and quality of the nucleic acids are sufficient for most PCR and RT-PCR applications, the process is too slow for high-throughput applications. A high-throughput DNA extraction method developed by Xin et al. (BioTechniques 34:820-826) for the analysis of cotton, sorghum, and other plants was evaluated as an alternative DNA extraction method for possible use in marker-assisted sugarcane breeding program. In this procedure, a small piece (about 30 mm2) of tissue from the youngest fully expanded leaf is excised and placed into a well of a 96-well plate containing a denaturing buffer (100 mM NaOH/2% Tween-20). The plate is sealed and placed on a PCR thermal cycler. After incubation at 95oC for 10 minutes, the plate is placed on ice for three minutes and spun at 3000 rpm for 1 minute. Fifty microliters of a neutralization buffer (100 mM Tris-HCl/2 mM EDTA) are then added to each well and the plate re-sealed with aluminum sealing tape. PCR amplifications of the HT products were comparable to those of the CTAB-Beadbeater method. Storage evaluations from the two methods of isolation also suggest that the HT-extracted nucleic acids can be stored in a refrigerator or freezer for later use with results from the stored material remaining consistent over time. With the HT method, a technician can process approximately 600 DNA samples per day, a ten-fold increase over the CTAB Beadbeater method of isolation. In addition the HT method uses neither organic solvent nor toxic compound.