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An improved semi-automated rapid method of extracting genomic DNA for molecular marker analysis in cocoa, Theobroma cacao L.

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Abstract
DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30–60% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4°C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/β-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of $0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.
Keywords
SSR, high-throughput, cocoa, DNA extraction, ball bearing

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Chicago
Bhattacharjee, Ranjana, Maria Kolesnikova-Allen, Peter Aikpokpodion, Sunday Taiwo, and Ivan Ingelbrecht. 2004. “An Improved Semi-automated Rapid Method of Extracting Genomic DNA for Molecular Marker Analysis in Cocoa, Theobroma Cacao L.” Plant Molecular Biology Reporter 22 (4): 435–436.
APA
Bhattacharjee, R., Kolesnikova-Allen, M., Aikpokpodion, P., Taiwo, S., & Ingelbrecht, I. (2004). An improved semi-automated rapid method of extracting genomic DNA for molecular marker analysis in cocoa, Theobroma cacao L. PLANT MOLECULAR BIOLOGY REPORTER, 22(4), 435–436.
Vancouver
1.
Bhattacharjee R, Kolesnikova-Allen M, Aikpokpodion P, Taiwo S, Ingelbrecht I. An improved semi-automated rapid method of extracting genomic DNA for molecular marker analysis in cocoa, Theobroma cacao L. PLANT MOLECULAR BIOLOGY REPORTER. 2004;22(4):435–6.
MLA
Bhattacharjee, Ranjana, Maria Kolesnikova-Allen, Peter Aikpokpodion, et al. “An Improved Semi-automated Rapid Method of Extracting Genomic DNA for Molecular Marker Analysis in Cocoa, Theobroma Cacao L.” PLANT MOLECULAR BIOLOGY REPORTER 22.4 (2004): 435–436. Print.
@article{1991532,
  abstract     = {DNA extraction is a time-consuming and expensive component of molecular marker analysis, constituting about 30--60\% of the total time required for sample processing. Furthermore, the procedure for extracting high-quality DNA from tree species such as cocoa differs from extraction protocols suitable for other crop plants. This is accompanied by problems in collecting leaf tissues from field-grown cocoa trees, where storage facilities are not available and where transporting samples to laboratory for immediate refrigeration is usually impossible. We preserved cocoa leaf tissues in the field in an NaCl-CTAB-azide solution (as described in Rogstad, 1992), which did not require immediate refrigeration. This method also allowed preservation of leaf tissues for a few days during transportation and protected leaf tissues from bacterial and fungal attacks. Once transported to the laboratory, the samples were stored at 4{\textdegree}C for almost 1 y. To isolate good-quality DNA from stored leaf tissues, a rapid semiautomated and relatively high-throughput protocol was established. The procedure followed a modified CTAB/\ensuremath{\beta}-mercaptoethanol method of DNA extraction in a 96-well plate, and an automated system (i.e., GenoGrinder 2000) was used to grind the leaf tissues. The quality of DNA was not affected by long storage, and the quantity obtained per sample was adequate for about 1000 PCR reactions. Thus, this method allowed isolation of about 200 samples per day at a cost of \$0.60 per sample and is a relatively high-throughput, low-cost extraction compared with conventional methods that use manual grinding and/or expensive kits.},
  author       = {Bhattacharjee, Ranjana and Kolesnikova-Allen, Maria and Aikpokpodion, Peter and Taiwo, Sunday and Ingelbrecht, Ivan},
  issn         = {0735-9640},
  journal      = {PLANT MOLECULAR BIOLOGY REPORTER},
  keyword      = {SSR,high-throughput,cocoa,DNA extraction,ball bearing},
  language     = {eng},
  number       = {4},
  pages        = {435--436},
  title        = {An improved semi-automated rapid method of extracting genomic DNA for molecular marker analysis in cocoa, Theobroma cacao L.},
  url          = {http://dx.doi.org/10.1007/BF02772686},
  volume       = {22},
  year         = {2004},
}

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