High-throughput genotyping of mannose-binding lectin variants using high-resolution DNA-melting analysis.
High Resolution Melting Analysis (HRMA) is a rapid and sensitive method for single nucleotide polymorphism (SNP) analysis. In the present study we present a novel HRMA assay to detect three SNPs in close proximity of each other in the first exon of the gene encoding mannose-binding lectin (MBL), a key molecule of innate immunity. These SNPs have been selected for their known biological and clinical relevance. The three SNPs in MBL2 were simultaneously determined in sixty-nine human DNA samples using HRMA and a single non-fluorescent melting probe, without any post-PCR processing of samples. Combining analyses from amplicon melting and probe melting, we have been able to discriminate ten exon 1 MBL2 genotypes with HRMA, making it a suitable tool for MBL genotyping. A second HRMA assay is presented to detect a relevant polymorphism (Y/X SNP) in the MBL2 promoter region. In conclusion, HRMA is a closed tube assay that is easy to setup and lends itself perfectly for high throughput genotyping of MBL2 variants. The present study thereby facilitates further clinical studies into the role of MBL in inflammatory and infectious disease.
Vossen
Rolf H A M
RH
Leiden Genome Technology Center, Human and Clinical Genetics, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands.
van Duijn
Martine
M
Daha
Mohamed R
MR
den Dunnen
Johan T
JT
Roos
Anja
A
D005838:Genotype; D006801:Humans; D037601:Mannose-Binding Lectin / Q000235:genetics*; D050505:Mutant Proteins / Q000235:genetics*; D009691:Nucleic Acid Denaturation / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA / Q000379:methods*
C445619:MBL2 protein, human; D037601:Mannose-Binding Lectin; D050505:Mutant Proteins