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Title: Asymmetric single-strand polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants

Author
item RODRIQUEZ, FLOR - University Of Wisconsin
item CAI, DANYING - Zhejiang Academy Of Agricultural Sciences
item TENG, YUANWEN - Zhejiang Academy Of Agricultural Sciences
item Spooner, David

Submitted to: American Journal of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/29/2011
Publication Date: 8/16/2011
Citation: Rodriquez, F., Cai, D., Teng, Y., Spooner, D.M. 2011. Asymmetric single-strand polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants. American Journal of Botany. 98:1061-1067.

Interpretive Summary: Wild and cultivated potatoes, technically grouped in the genus Solanum, section Petota, are very similar. Their formal classification into species is difficult, and there are many conflicting publications (taxonomies) that recognize different numbers of species. Molecular data from DNA sequences provides a powerful and very useful method to help determine both the knowledge of what is a species and to determine how these species are interrelated to each other. However, obtaining DNA sequences can have many problems, especially in plants that have complex difference in DNA types among individuals or lines, technically termed DNA polymorphism. This study develops a procedure to solve this problem in a way that is both relatively inexpensive and that increases the accuracy of obtaining different DNA sequences, technically termed asymmetric single-strand polymorphism.

Technical Abstract: We needed to obtain an alternative to conventional cloning to generate high-quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies in potato, to save time and money and to avoid problems typically encountered in cloning. We tested a variety of SSCP protocols to include purified and unpurified symmetric and asymmetric PCR, loading buffers, and electrophoresis conditions (buffers, matrix, running time, temperature); compared these to cloning with the same samples; and analyzed the sequences with maximum likelihood. Our optimized protocol use asymmetric PCR, with the majority of the samples run in polyacrilamide gel electrophoresis (PAGE), and was able to consistently able to separate PCR products from 450-1200 (1500) bp. Asymmetrical single-strand polymorphism provides is a greatly improved technique for isolating allelic variants of highly heterozygous individuals relative to cloning, with its greatest applications in obtaining DNA sequences for allopolyploids. It is much cheaper and eliminates two common problems encountered in cloning: PCR recombination and heteroduplex fixation.