PCR-based genotyping was conducted with a primer pair (common forward primer: 5'-CCTGTCCCAAGGACTGGCATC-3'; reverse primer for L100L wild-type: 5'-CAGGGACAAGGGAGCTCTTCA-3'; reverse primer for L100P mutants: 5'- CandgenomicGAGCTCTTCG-3') anDNAextractedA extracted from tail biopsies using Hot Sodium Hydroxide and Tris (HotSHOT) method [ 30].
Genotyping was conducted using single-base chain extension assays modified by GlaxoSmithKline (Research Triangle Park, NC, USA), TaqMan SNP assays (Applied Biosystems, Foster City, CA, USA), GoldenGate and Infinium genotyping assays (Illumina, San Diego, CA, USA), the KASPar SNP genotyping system (LGC Genomics, Hoddesdon, UK), and Sanger sequencing.
Additional SNP genotyping was conducted using the Mismatch Amplification Mutation Assay [MAMA] [ 29], which is based on allele-specific PCR kinetics [ 48], enhanced by penultimate mismatch primer design [ 29, 49].
Genotyping was conducted by the Vanderbilt DNA Resources Core using the mid-throughput Sequenom® genotyping platform (rs1333049, rs3135388 and rs17234657; genotyping efficiency 98.4 100%), which is based on a single base primer extension reaction coupled with mass spectrometry, or using a TaqMan assay (rs6457620 and rs2200733; genotyping efficiency 99.4% and 99.0%, respectively).
SSR genotyping was conducted by YK.
Genotyping was conducted at Diversity Arrays Technology Pty Ltd. (DArT P/L), Australia.
For SNP and functional markers, multiplex genotyping was conducted using the SNPstream system (Beckman Coulter, California, USA).
Replication genotyping was conducted at the Murdoch Children's Research Institute Sequenom Platform Facility.
SNaPshot™ genotyping was conducted in London and Cambridge following slightly differing protocols.
All genotyping was conducted at the Genotype Shared Resource (GSR) at the Mayo Clinic, Rochester, MN.
