Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes
2012 (English)In: Molecular Ecology Resources, ISSN 1755-098X, Vol. 12, no 1, 128-135 p.Article in journal (Refereed) Published
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 1513 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.
Place, publisher, year, edition, pages
2012. Vol. 12, no 1, 128-135 p.
allele-specific primer, amplification refractory mutation system, low-copy nuclear gene, primer design
IdentifiersURN: urn:nbn:se:uu:diva-169231DOI: 10.1111/j.1755-0998.2011.03070.xISI: 000298585000015OAI: oai:DiVA.org:uu-169231DiVA: diva2:507178