The MC1R gene encodes a protein with key regulatory functions in the melanin 
synthesis. A multiplex PCR and a multiplex single base extension protocol were 
established for genotyping six exonic MC1R variations highly penetrant for red 
hair (R), four exonic MC1R variations weakly penetrant for red hair (r), two 
frameshift variations highly penetrant for red hair (R) and three variations in 
the promoter region. We genotyped 600 individuals from Denmark using either CE 
or MALDI-TOF MS as the detection platform. A total of 62 individuals were 
genotyped R/R and among the 62 individuals, 57 had red hair and five had blond 
hair colour. Two different R alleles may be located in cis (RR/-) position or 
trans (R/R) position, and the phenotype associated with RR/- and R/R may be 
different. Two allele-specific PCRs were established with primers targeting the 
-G445A variation in the MC1R promoter and the allele-specific PCR products were 
used in the multiplex single base extension assay. In all 62 individuals, the 
MC1R variants were situated in trans position. Another 18 individuals with red 
hair colour were either genotyped R/- or R/r, suggesting that other genes 
influence hair colour.