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Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes

Journal article
Authors Anne-Cathrine Scheen
Bernard E. Pfeil
Anna Petri
N. Heidari
Stephan Nylinder
Bengt Oxelman
Published in Molecular Ecology Resources
Volume 12
Issue 1
Pages 128-135
ISSN 1755-0998
Publication year 2012
Published at Department of Biological and Environmental Sciences
Pages 128-135
Language en
Keywords allele-specific primer; amplification refractory mutation system; low-copy nuclear gene; primer design
Subject categories Biological Systematics, Evolutionary Biology


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.

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