Present address: Museum of Archaeology, University of Stavanger, NO-4036 Stavanger, Norway.
Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes
Article first published online: 21 SEP 2011
© 2011 Blackwell Publishing Ltd
Molecular Ecology Resources
Volume 12, Issue 1, pages 128–135, January 2012
How to Cite
SCHEEN, A.-C., PFEIL, B. E., PETRI, A., HEIDARI, N., NYLINDER, S. and OXELMAN, B. (2012), Use of allele-specific sequencing primers is an efficient alternative to PCR subcloning of low-copy nuclear genes. Molecular Ecology Resources, 12: 128–135. doi: 10.1111/j.1755-0998.2011.03070.x
- Issue published online: 16 DEC 2011
- Article first published online: 21 SEP 2011
- Received 28 March 2011; revision received 29 July 2011; accepted 15 August 2011
- allele-specific primer;
- amplification refractory mutation system;
- low-copy nuclear gene;
- primer design
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.