Improved Ligation Specificity with Chemically Modified Ligation Components
Poster Oct 04, 2012
Sabrina Shore, Alexandre Lebedev, Elena Hidalgo Ashrafi, Gerald Zon, Natasha Paul, Richard Hogrefe
Ligases are gaining utility in molecular biology applications, such as nucleotide sequence detection, single nucleotide polymorphism (SNP) detection, protein detection and “next generation” sequencing by ligation. With the increased demand for DNA ligases in the field of biotechnology, comes increased demand for ligation fidelity. Described approaches to improved ligation fidelity include ligases from different biological sources, point mutations of key amino acid residues within the ligase, modified reaction conditions and addition of crowding reagents, such as PEG. Although most approaches to improved ligation fidelity have focused on the ligase itself, further improvements are needed and may be attainable by a different approach. Herein a strategy to improve the discrimination between matched and mismatched targets is described which employs chemical modification to the nucleic acid components of the reaction, such as the donor probe, the acceptor probe and the ATP cofactor. The results demonstrate that chemically modified components increase the stringency of DNA ligase-mediated nucleic acid detection, providing a unique approach for SNP genotyping.
Despite the developments in conventional PCR, the complexity of multiplex Real Time PCR is still limited due to the lack of sufficient detection channels. To achieve high-end multiplexing capacity on standard Real Time PCR machines, Anapa Biotech has developed the MeltPlex® technology (see box on right).READ MORE
Genome-wide association studies (GWAS) have identified more than 100 genetic loci associated with type 2 diabetes. The majority of these are located in the intergenic or intragenic regions suggesting that the implicated variants may alter chromatin conformation. This, in turn, is likely to influence the expression of nearby or more remotely located genes to alter beta cell function. At present, however, detailed molecular and functional analyses are still lacking for most of these variants. We recently analysed one of these loci and mapped five causal variants in an islet-specific enhancer cluster within the STARD10 gene locus. Here, we aimed to understand how these causal variants influence b-cell function by alteration of the chromatin structure of enhancer clusterREAD MORE