Rapid genotyping of MBL2 gene mutations using real-time PCR with fluorescent hybridisation probes.
In this study, we describe a real-time polymerase chain reaction (PCR) for genotyping all known polymorphisms of the human mannose-binding lectin 2 (MBL2) gene. These comprised two variations in the 5' regulatory region at positions -550 (H/L) and -221 (X/Y), one in the 5' untranslated sequence at position +4 (P/Q) and three structural mutations within exon 1 at codons 52, 54, and 57, also known as the D, B and C variants, respectively. Three reactions with two different conditions were sufficient to genotype one individual unambiguously. The three mutations in exon 1 were detected in one capillary using a sensor probe covering the three mutations, whereas amplification of the variants located upstream of the coding sequence was performed in only two reactions. Single colour detection was used for detection of the (H/L) polymorphism and multiplexing by dual colour probes was used for simultaneous genotyping of (X/Y) and (P/Q). The reliability of the system was evaluated by comparison with a conventional PCR method with sequence-specific primers (PCR-SSP). For this study, 100 individuals of Danish and 30 of African descent were analysed, and the genotypes obtained were concordant in all cases. This new method is rapid and provides reliable results without ambiguities.
Steffensen
R
R
Regional Centre for Blood Transfusion and Clinical Immunology, Aalborg Hospital, 9000 Alborg, Denmark. rudi@aas.nja.dk
Hoffmann
K
K
Varming
K
K
D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005838:Genotype; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D037601:Mannose-Binding Lectin / Q000031:analogs & derivatives* / Q000235:genetics*; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D011401:Promoter Regions, Genetic
D017931:DNA Primers; C445619:MBL2 protein, human; D037601:Mannose-Binding Lectin