title: Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes.
abstract: Direct Sanger sequencing of polymerase chain reaction (PCR)-amplified nuclear genes leads to polymorphic sequences when allelic variation is present. To overcome this problem, most researchers subclone the PCR products to separate alleles. An alternative is to directly sequence the separate alleles using allele-specific primers. We tested two methods to enhance the specificity of allele-specific primers for use in direct sequencing: using short primers and amplification refractory mutation system (ARMS) technique. By shortening the allele-specific primer to 15-13 nucleotides, the single mismatch in the ultimate base of the primer is enough to hinder the amplification of the nontarget allele in direct sequencing and recover only the targeted allele at high accuracy. The deliberate addition of a second mismatch, as implemented in the ARMS technique, was less successful and seems better suited for allele-specific amplification in regular PCR rather than in direct sequencing.
authors.affiliation: Department of Plant and Environmental Sciences, University of Gothenburg, Box 461, SE 405 30 Gothenburg, Sweden. anne-cathrine.scheen@uis.no
authors.forename: Anne-Cathrine
authors.initials: AC
authors.lastname: Scheen
authors.forename: Bernard E
authors.initials: BE
authors.lastname: Pfeil
authors.forename: Anna
authors.initials: A
authors.lastname: Petri
authors.forename: Nahid
authors.initials: N
authors.lastname: Heidari
authors.forename: Stephan
authors.initials: S
authors.lastname: Nylinder
authors.forename: Bengt
authors.initials: B
authors.lastname: Oxelman
mesh_terms: D000483:Alleles; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics*; D031312:Fumaria / Q000235:genetics*; D018628:Gene Dosage; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013045:Species Specificity
chemical_list: D017931:DNA Primers