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till bradley j.; jankowicz-cieslak joanna; huynh owen a.; beshir mayada m.; laport robert g.; hofinger bernhard j. - low-cost methods for molecular characterization of mutant plants

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

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Lingua: Inglese


Pubblicazione: 10/2016
Edizione: Softcover reprint of the original 1st ed. 2015


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


2             Health and Safety Considerations

               2.1 Guidelines

               2.2 Preparation of a home-made chemical spill kit


3             Sample Collection and Storage

3.1 Background

3.2 Materials

3.3 Methods


4             Low-Cost DNA Extraction

               4.1 Materials

   4.2 Methods

   4.3 Alternative buffers for DNA extraction


5             PCR Amplification for Low-Cost Mutation Discovery

               5.1 Materials

               5.2 Methods


6             Enzymatic Mismatch Cleavage and Agarose Gel Evaluation of Samples

6.1 Materials

6.2 Methods


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    

Altre Informazioni



Condizione: Nuovo
Dimensioni: 235 x 155 mm Ø 97 gr
Formato: Brossura
Illustration Notes:6 Illustrations, black and white
Pagine Arabe: 35
Pagine Romane: x

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