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Multiplex SNP‐SCALE: a cost‐effective medium‐throughput single nucleotide polymorphism genotyping method
Author(s) -
KENTA T.,
GRATTEN J.,
HAIGH N. S.,
HINTEN G. N.,
SLATE J.,
BUTLIN R. K.,
BURKE T.
Publication year - 2008
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.1755-0998.2008.02190.x
Subject(s) - biology , genotyping , molecular inversion probe , snp genotyping , multiplex , snp , single nucleotide polymorphism , multiplex polymerase chain reaction , polymerase chain reaction optimization , polymerase chain reaction , primer (cosmetics) , genetics , computational biology , microbiology and biotechnology , genotype , gene , chemistry , organic chemistry
We describe a convenient, cost‐effective and flexible medium‐throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP‐SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP‐SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four‐dye system, (iii) much‐reduced (2‐µL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig‐tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP‐SCALE P rimer D esigner ) that automatically designs suitable SNP‐SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost‐ and time‐effective for medium‐scale evolutionary and ecological projects involving 10s to 100s of loci.