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Table 1.

Primer and hybridization probe sequences, conditions, and annealing temperature of real-time RT-PCR

MarkerPrimer/hybridization probe sequenceAmplicon (bp)Annealing temperature (°C)MgCl2 concentration (mmol/L)
Porphobilinogen deaminase     
    Forward 5′-TGCAGGCTACCATCCATGTCCCTGC-3′ 187 65 4 
    Reverse 5′-AGCTGCCGTGCAACATCCAGGATGT-3′    
    Probe 5′-CGTGGAATG TTACGAGCAGTGATGCCTACC-Fluo-3′    
    Probe 5′-LCRed-640-TGTGGGTCATCCTCAGGGCCATCTTC-Pho-3′    
Tyrosinase     
    Forward 5′-GTCTTTATGCAATGGAACGC-3′ 207 60 3 
    Reverse 5′-GCTATCCCAGTAAGTGGACT-3′    
    Probe 5′-GCGTAATCCTGGAAACCATGACAAA-Fluo-3′    
    Probe 5′-LCRed-640-CACAACCCCAAGGCTCCCCTCTTC-Pho-3′    
MelanA/MART1     
    Forward 5′-CACTCTTACACCACGGCTGA-3′ 300 65 4 
    Reverse 5′-AGGTGAATAAGGTGGTGGTGA-3′    
    Probe 5′-GCTGTCCCGATGATCAAACCCTTC-Fluo-3′    
    Probe 5′-LCRed-640-TGTGGGCATCTTCTTGTTAAGGCACA-Pho-3′    
MarkerPrimer/hybridization probe sequenceAmplicon (bp)Annealing temperature (°C)MgCl2 concentration (mmol/L)
Porphobilinogen deaminase     
    Forward 5′-TGCAGGCTACCATCCATGTCCCTGC-3′ 187 65 4 
    Reverse 5′-AGCTGCCGTGCAACATCCAGGATGT-3′    
    Probe 5′-CGTGGAATG TTACGAGCAGTGATGCCTACC-Fluo-3′    
    Probe 5′-LCRed-640-TGTGGGTCATCCTCAGGGCCATCTTC-Pho-3′    
Tyrosinase     
    Forward 5′-GTCTTTATGCAATGGAACGC-3′ 207 60 3 
    Reverse 5′-GCTATCCCAGTAAGTGGACT-3′    
    Probe 5′-GCGTAATCCTGGAAACCATGACAAA-Fluo-3′    
    Probe 5′-LCRed-640-CACAACCCCAAGGCTCCCCTCTTC-Pho-3′    
MelanA/MART1     
    Forward 5′-CACTCTTACACCACGGCTGA-3′ 300 65 4 
    Reverse 5′-AGGTGAATAAGGTGGTGGTGA-3′    
    Probe 5′-GCTGTCCCGATGATCAAACCCTTC-Fluo-3′    
    Probe 5′-LCRed-640-TGTGGGCATCTTCTTGTTAAGGCACA-Pho-3′    
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