Abstract
Standard agarose gel electrophoresis is a quick method for the evaluation of the quality and quantity of DNA. This chapter provides examples of genomic DNA produced using the low-cost extraction protocol, PCR amplification using the extracted genomic DNA, and enzymatic mismatch cleavage of PCR products with crude celery juice extract and weed juice extract to detect mutations.
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8.1 Quality of Genomic DNA Obtained by Silica Powder-Based DNA Extraction Method
While spectrophotometric approaches (e.g., Nanodrop) provide a quick and accurate measure of DNA concentration and protein contamination, and fluorometric methods (e.g., Qubit) provide high sensitivity, it is advisable that when optimizing the DNA extraction protocol, samples are also run on a traditional agarose gel. This allows an estimation of DNA concentration (relative to concentration standards such as lambda DNA, Till et al. 2007), the extent of RNA carryover, as well as an estimation of the extent of DNA degradation, something which cannot be easily determined from the other two techniques (Figs. 8.1 and 8.2). Furthermore, chaotropic salts can lower the accuracy of spectrophotometric methods. Reducing sample degradation may be a key optimization step for some species. Alternative buffers can be employed to limit degradation and the copurification of secondary metabolites that can inhibit downstream molecular assays (see Sect. 8.2).
8.2 Quality of Genomic DNA Obtained by Silica Powder-Based DNA Extraction Method Using Alternative Buffers
Alternative buffers can be used to extract DNA from tissues or species where the use of the standard buffer produces low-quality DNA. Tissue from Sorghum bicolor and grapevine (Vitis vinifera) were used to compare different buffer compositions (Tables 8.1 and 8.2).
Quality and quantity of genomic DNA using different buffers were assayed by native agarose gel electrophoresis (Fig. 8.3).
8.2.1 Summary
High yield and high quality genomic DNA can be recovered from sorghum using a simple lysis buffer. This buffer, however, is not suitable for grapevine DNA extraction and alternative buffers are required to recover high molecular weight DNA, albeit at a lower concentration than can be achieved from sorghum samples. This suggests further parameter changes can be made to increase yields.
8.3 Example of PCR Products Using TILLING Primers with Source Genomic DNA from a Commercial Kit and Low-Cost Silica Method
High quality and quantity of gene-specific PCR products are produced in reactions where source genomic DNA is extracted using either commercial kits or the low-cost silica method (Fig. 8.4).
8.4 Example of Low-Cost Agarose Gel-Based TILLING Assays for the Discovery of Induced Point Mutations
PCR products are the substrate for enzymatic mismatch cleavage assays for mutation discovery. Agarose gels provide a low-cost platform for mutation discovery using self-extracted enzymes (Fig. 8.5).
8.5 Example of Enzyme Activity Recovered from Weeds Compared to Crude Celery Juice Extract
The recovery of proteins from collected weeds versus celery is listed in Table 8.3. This was used to prepare reaction mixes (Table 8.4) to test for enzymatic activity. Samples were evaluated via standard agarose gel electrophoresis (Fig. 8.6).
8.5.1 Summary
Crude enzyme extracts of weeds show a similar activity to that of celery extract for the cleavage of single nucleotide polymorphisms. The per unit activity, however, was lower than for CEL I, likely owing to the co-precipitation of other plant proteins in weeds, presumably including a larger amount of RuBisCO. This limitation can be overcome through the use of a simple centrifugation-based protein concentration step. Using this protocol, 150 ml of weed extract produces sufficient enzyme for approximately 2,000 reactions.
References
Hofinger BJ, Huynh OA, Jankowicz-Cieslak J, Müller A, Otto I, Kumlehn J, Till BJ (2013) Validation of doubled haploid plants by enzymatic mismatch cleavage. Plant Methods 9(1):43
Till BJ, Burtner C, Comai L, Henikoff S (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res 32:2632–2641
Till BJ, Cooper CJ, Tai TH, Colowit P, Greene EA, Henikoff S, Comai L (2007) Discovery of chemically induced mutations in rice by TILLING. BMC Plant Biol 7:19
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Till, B.J., Jankowicz-Cieslak, J., Huynh, O.A., Beshir, M.M., Laport, R.G., Hofinger, B.J. (2015). Example Data. In: Low-Cost Methods for Molecular Characterization of Mutant Plants. Springer, Cham. https://doi.org/10.1007/978-3-319-16259-1_8
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DOI: https://doi.org/10.1007/978-3-319-16259-1_8
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