Abstract
Large-scale use of molecular markers in plant breeding is limited by the throughput capacity for genotyping. DNA polymorphisms can be detected in denaturing polyacrylamide gels indirectly by nucleotide labeling or directly by staining. Fluorescent-labeling or radiolabeling requires sophisticated infrastructure not always available in developing countries. We present an improved low-cost method for silver staining and compare it to 2 other methods for their ability to detect simple sequence repeat polymorphisms in denaturing polyacrylamide gels bound to glass plates. The 3 procedures differed in their requirement for an oxidation pretreatment, preexposure with formaldehyde during silver nitrate impregnation, inclusion of silver thiosulfate, and by their replacement of sodium carbonate for sodium hydroxide to establish alkaline conditions for silver ion reduction. All methods detected the same banding pattern and alleles. However, important differences in sensitivity, contrast, and background were observed. Two methods gave superior sensitivity, detecting down to 1 μL of loaded amplification products. Our improved method gave lower backgrounds and allowed reutilization of staining solutions. The use of thin (<1 mm) denaturing sequencing gels allows genotyping of 60–96 samples within 4 h. Use of smaller loading sample volumes and reutilization of staining solutions further reduced costs.
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Abbreviations
- SSR:
-
simple sequence repeat
- TBE:
-
tris borate ethylenediaminetetracetic acid
- TEMED:
-
tetramethylethylenediamine
References
Bassam BJ, Caetano-Anollées G, and Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 196: 80–83.
Beidler JL, Hilliard PR, and Rill RL (1982) Ultrasensitive staining of nucleic acids with silver. Anal Biochem 126: 374–380.
Blum, H, Beier H, and Gross HJ (1987) Improved silver staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93–99.
Christensen M, Sunde L, Bolund L, and Orntoft TF (1999) Comparison of three methods of microsatellite detection. Scand J Clin Lab Invest 59: 167–178.
Comincini S, Leone P, Redaelli L, DeGiuli L, Zhang Y, and Ferretti L (1995) Characterization of bovine microsatellites by silver staining. J Anim Breed Genet 112: 415–420.
Doyle JJ and Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12: 13–15.
Herring AJ, Inglis NF, Ojeh CK, Snodgrass DR, and Menzies JD (1982) Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels. J Clin Microbiology 16: 473–477.
Lagoda PJL, Dambier D, Grapin A, Baurens FC, Lanaud C, and Noyer JL (1998a) Nonradioactive sequence-tagged microsatellite site analyses: a method transferable to the tropics. Electrophoresis 19: 152–157.
Lagoda PJL, Noyer JL, Dabier D, Baurens FC, Grapin A, and Lanaud C (1998b) Sequence tagged microsatellite site (STMS) markers in the Musaceae. Molecular Ecology 7: 657–666.
Merril CR, Goldman D, Sedman SA, and Ebert MH (1981) Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science 211: 1437–1438.
Mitchell SE, Kresovich S, Jester CA, Hernandez CJ, and Szwec-McFadden AK (1997) Application of multiplex PCR and fluorescence-based, semi-automated allele sizing technology for genotyping plant genetic resources. Crop Sci. 37: 617–624.
Sanguinetti CJ, Dias Neto E, and Simpson AJG (1994) Rapid silver staining and recovery of PCR products separated on polyacrylamide gels. Biotechniques 17: 915–919.
Santos FR, Pena SDJ, and Epplen JT (1993) Genetic and population study of a Y-linked tetranucleotide repeat DNA polymorphism with a simple non-isotopic technique. Human Genet 90: 655–656.
Sommerville LL and Wang K (1981) The ultrasensitive silver protein stain also detects nanograms of nucleic acids. Biochem Biophys Res Commun 102: 530–548.
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Creste, S., Neto, A.T. & Figueira, A. Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining. Plant Mol Biol Rep 19, 299–306 (2001). https://doi.org/10.1007/BF02772828
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DOI: https://doi.org/10.1007/BF02772828