Rapid genotyping of single nucleotide polymorphisms using novel minor groove binding DNA oligonucleotides (MGB probes).
Novel fluorescent oligonucleotides that contain a 3' minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5' nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554-559, 2002.
de Kok
Jacques B
JB
Department of Clinical Chemistry, University Medical Centre, Nijmegen, The Netherlands. J.dekok@ckcl.azn.nl
Wiegerinck
Erwin T G
ET
Giesendorf
Belinda A J
BA
Swinkels
Dorine W
DW
D000483:Alleles; D003062:Codon / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods; D004277:DNA, Single-Stranded / Q000302:isolation & purification / Q000378:metabolism*; D005805:Genes, MHC Class I / Q000235:genetics; D005838:Genotype; D006680:HLA Antigens / Q000235:genetics; D006432:Hemochromatosis / Q000235:genetics; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins; D008875:Middle Aged; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods; D015345:Oligonucleotide Probes / Q000302:isolation & purification / Q000378:metabolism*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D013403:Sugar Phosphates / Q000737:chemistry / Q000378:metabolism
D003062:Codon; D004277:DNA, Single-Stranded; C103673:HFE protein, human; D006680:HLA Antigens; D000071020:Hemochromatosis Protein; D015395:Histocompatibility Antigens Class I; D008565:Membrane Proteins; D015345:Oligonucleotide Probes; D013403:Sugar Phosphates