Low-Cost Methods for Molecular Characterization of Mutant Plants Tissue Desiccation, DNA Extraction and Mutation Discovery: Protocols

This book offers low-cost and rapid molecular assays for the characterization of mutant plant germplasm. Detailed protocols are provided for the desiccation of plant tissues; the extraction of high-quality DNA for downstream applications; the extraction of single-strand-specific nucleases for single nucleotide polymorphism; and small insertion/deletion discovery using standard agarose gel electrophoresis. The methods described can be applied in any laboratory equipped for basic molecular biology and do away with the need for expensive freezers and toxic organic compounds. With the appropriate validation of sample quality and longevity, they can provide sufficient DNA for a variety of molecular applications, such as marker studies and TILLING, at approximately one tenth of the cost per sample when compared to commercial kits.

1             Introduction

1.1 Background

1.2 Methods used to isolate genomic DNA from plant tissues

1.3 Methods for the discovery and characterization of induced and natural nucleotide variation in plant genomes

References

2             Health and Safety Considerations

               2.1 Guidelines

               2.2 Preparation of a home-made chemical spill kit

References

3             Sample Collection and Storage

3.1 Background

3.2 Materials

       References

4             Low-Cost DNA Extraction

               4.1 Materials

   4.2 Methods

   4.3 Alternative buffers for DNA extraction

   References

5             PCR Amplification for Low-Cost Mutation Discovery

               5.1 Materials

               5.2 Methods

               References

6             Enzymatic Mismatch Cleavage and Agarose Gel Evaluation of Samples

6.2 Methods

References

7          Alternative Enzymology for Mismatch Cleavage for TILLING and Ecotilling: Extraction of Enzymes form Common Weedy Plants

7.1 Materials

7.2 Methods

8          Example Data

8.1 Quality of genomic DNA obtained by silica powder-based DNA extraction method

8.2 Quality of genomic DNA obtained by silica powder-based DNA extraction using alternative buffers method

8.3 Example of PCR products using TILLING primers with source genomic DNA from Qiagen kits and low-cost silica method

8.4 Example of low-cost agarose gel based TILLING assays for the discovery of induced point mutations

8.5 Example of enzyme activity recorded from weeds compared to crude celery juice extract

9             Conclusions