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Asymmetric single‐strand conformation polymorphism: An accurate and cost‐effective method to amplify and sequence allelic variants
Author(s) -
Rodríguez Flor,
Cai Danying,
Teng Yuanwen,
Spooner David
Publication year - 2011
Publication title -
american journal of botany
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.218
H-Index - 151
eISSN - 1537-2197
pISSN - 0002-9122
DOI - 10.3732/ajb.1000251
Subject(s) - biology , single strand conformation polymorphism , heteroduplex , cloning (programming) , genetics , computational biology , dna , polymerase chain reaction , gene , computer science , programming language
• Premise of the study: An efficient alternative strategy to conventional cloning was needed to generate high‐quality DNA sequences from a variety of nuclear orthologs for phylogenetic studies. This method would facilitate studies and minimize technical problems typically encountered in cloning methodologies. • Methods: We tested a variety of single‐strand conformation polymorphism (SSCP) protocols including purified and unpurified symmetric and asymmetric PCR, loading buffers, and electrophoresis conditions (buffers, matrix, running time, temperature). Results obtained from direct SSCP band sequencing were compared to those obtained from cloning. • Key results: Our optimized protocol uses asymmetric PCR, with the majority of the samples run in polyacrylamide gel electrophoresis (PAGE). It consistently separated PCR products from 450 to 1200 bp. • Conclusions: Asymmetric PCR single‐strand conformation polymorphism is an efficient alternative technique for isolating allelic variants of highly heterozygous individuals, with its greatest applications in sequencing allopolyploids. It eliminates two common problems encountered in cloning: PCR recombination and heteroduplex fixation. In addition, our protocol greatly lowers costs and time associated with procedures.