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Use of allele‐specific sequencing primers is an efficient alternative to PCR subcloning of low‐copy nuclear genes
Author(s) -
SCHEEN ANNECATHRINE,
PFEIL BERNARD E.,
PETRI ANNA,
HEIDARI NAHID,
NYLINDER STEPHAN,
OXELMAN BENGT
Publication year - 2012
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/j.1755-0998.2011.03070.x
Subject(s) - biology , sanger sequencing , subcloning , genetics , allele , variants of pcr , primer (cosmetics) , polymerase chain reaction , primer dimer , gene , dna sequencing , multiple displacement amplification , microbiology and biotechnology , multiplex polymerase chain reaction , plasmid , dna extraction , chemistry , organic chemistry
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.