title: Asymmetric single-strand conformation polymorphism: an accurate and cost-effective method to amplify and sequence allelic variants.
abstract: OBJECTIVE
  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.
  
  
  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.
authors: USDA, Agricultural Research Service, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, Wisconsin 53706-1590, USA.
authors: Flor
authors: F
authors: Rodríguez
authors: Danying
authors: D
authors: Cai
authors: Yuanwen
authors: Y
authors: Teng
authors: David
authors: D
authors: Spooner
mesh_terms: D000483:Alleles; D003362:Cost-Benefit Analysis; D009154:Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000191:economics* / Q000379:methods*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D018807:Polymorphism, Single-Stranded Conformational / Q000235:genetics*; D011123:Polyploidy; D017422:Sequence Analysis, DNA / Q000191:economics* / Q000379:methods*; D017384:Sequence Deletion / Q000235:genetics; D032322:Solanum / Q000235:genetics; D013045:Species Specificity