Detection of single sequence repeat polymorphisms in denaturing polyacrylamide sequencing gels by silver staining

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 inf...

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Bibliographic Details
Published in:Plant molecular biology reporter 2001-12, Vol.19 (4), p.299-306
Main Authors: Creste, S, Tulmann Neto, A, Figueira, A
Format: Article
Language:English
Subjects:
amplified fragment length polymorphism
Banding
cost effectiveness
cultivars
Deoxyribonucleic acid
Developing countries
differential staining
diploidy
DNA
DNA fingerprinting
Fluorescence
Gels
genetic markers
genetic polymorphism
Genotyping
Labeling
LDCs
leaves
methodology
microsatellite repeats
Musa
nucleotide sequences
Oxidation
Plant breeding
polyacrylamide gel electrophoresis
Pretreatment
Sensitivity
Silver
Silver nitrate
Silver thiosulfate
Sodium carbonate
Sodium hydroxide
Staining
tetraploidy
Thiosulfate
triploidy
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Summary: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 (
ISSN:0735-9640
1572-9818
DOI:10.1007/BF02772828