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

摘要

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 muL 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.