Development of the Ligation Detection Reaction (LDR)-TaqMan Assay

The LDR-TaqMan SNP genotyping method was elaborated and patented [Borodina et al., 2004; EP03019521]. The method is based on the ligation detection reaction (LDR), which is performed directly on genomic DNA. During the LDR, the biallelic state of an SNP locus is converted into the bimarker state of ligated detector oligonucleotides. The state of the markers is then determined by the 5'-nuclease assay (TaqMan) with universal fluorescent probes. The technology is sensitive, has high discrimination power, needs no locus-specific optimization and is applicable both for individual and multiplex SNP analyses. To transfer the technology to scientific partners, the following additional tasks were solved: a method for cost-effective synthesis of long (60-120nt) oligonucleotides with block structure was developed and patented [Borodina et al., 2003; PCT/EP 2004/003921]; a procedure for DNA isolation from plant material and saliva on home-made silica spin-columns was established [Borodina et al., 2003a]; enzymes required for the method (thermostable Pfu DNA ligase and hot start Taq DNA polymerase) were cloned and expressed. The LDR-TaqMan method was applied for SNP genotyping of human and Arabidopsis thaliana DNA samples: a kit for genotyping of 9 clinically important human SNPs was prepared and used for determination of allele frequencies of these SNPs in two East European populations (Ukrainians and Belorussians, 216 DNA samples) [Kozhekbaeva et al., 2004]; a 138-loci genotyping kit for Arabidopsis thaliana accessions Columbia (Col-0) and C24 was prepared and tested in the blind genotyping of 10 plants. A comparison of the LDR-TaqMan genotyping results with those obtained by conventional methods (sequencing, RFLP, SNaPshot, Amplifluor [Rickert et al., 2004]) demonstrated the efficiency and reliability of the LDR-TaqMan procedure. Ready-to-use genotyping kits (including enzymes, buffers and sets of detector oligonucleotides) were transferred to collaborators in Golm (Germany) and Moscow (Russia). %%%% Im Rahmen der Arbeit wurde die LDR-TaqMan SNP-Genotypisierungsmethode entwickelt und patentiert [Borodina et al., 2004; EP03019521]. Die Methode basiert auf der direkt mit genomischer DNA durchgefuhrten Ligationsdetektionsreaktion (LDR). Abhangig vom allelischen Zustand des SNP Locus wird eine korrespondierende Markersequenz in das Ligationsprodukt integriert. Die Markersequenz wird im folgenden 5'-Exonukleaseassay (TaqMan) durch universelle Sonden identifiziert. Die Methode ist sensitiv, hat ein hohes Diskriminierungsvermogen, bedarf keiner lokusspezifischen Optimierung und ist in Single- als auch Multiplexanalysen anwendbar. Um die Technologie unseren wissenschaftlichen Partnern zur Verfugung zu stellen, wurden folgende Aufgaben gelost: die kosteneffiziente Synthese langer (60 - 120nt) Oligonukleotide mit Blockstruktur [Borodina…