Malditof Ms For Snp Genotyping

Nucleic acids are more difficult to analyze under MALDI conditions than peptides, which had limited its application in this field. Because of their negatively charged phosphate backbone nucleic acids are susceptible to adduct formation. They form salt adducts with cations present in the surrounding medium (e.g., buffers). Adduct formation results in a broader distribution of the signal. For example, sodium ions attached to the analyte will cause an additional signal with a mass of plus 22 Da (23 for sodium minus 1 for an exchanged hydrogen). Consequently, adduct formation lowers sensitivity and analytical accuracy. In addition to strong adduct formation, nucleic acids also tend to fragment under MALDI conditions.

The main reaction contributing to DNA fragmentation under MALDI conditions is depurination. Depurination occurs through protonation of the nucleobases A or G (at position N7). The protonation induces polarization of the N-glycosidic bond between sugar and nucleobase. The result is nucleobase elimination. Subsequent to depuri-nation reactions, further fragmentation occurs via backbone cleavage.

Fig. 1 Box A illustrates the MassEXTEND1 primer extension concept for SNP genotyping. Spectra derived from a uniplex reaction are displayed for both homozygote cases and the heterozygote condition. Box B provides raw data for a highly multiplexed reaction for SNP genotyping following the same schema as outlined in A but employing multiple PCR and MassEXTEND1 primer in one reaction vessel.

Fig. 1 Box A illustrates the MassEXTEND1 primer extension concept for SNP genotyping. Spectra derived from a uniplex reaction are displayed for both homozygote cases and the heterozygote condition. Box B provides raw data for a highly multiplexed reaction for SNP genotyping following the same schema as outlined in A but employing multiple PCR and MassEXTEND1 primer in one reaction vessel.

The availability of favorable matrices for DNA analysis is an important factor for the success of MALDI-TOF MS in the field of DNA analysis. Especially, the introduction of 3-hydroxypicolinic acid (3-HPA) as matrix[1] is a cornerstone in this respect.

The most predominant method used for MALDI-TOF MS-based SNP genotyping relies on primer extension concepts—the so-called MassEXTEND1 assay.1-2-1 MassEXTEND1 assays combine primer extension with MALDI-TOF MS. The assay consists of a post-PCR primer extension reaction that is carried out in the presence of one or more dideoxynucleotides (ddNTPs) generating allele-specific terminated extension products. Primers are designed to anneal adjacent to the SNPs of interest. Depending on the SNP identity, extension products of different lengths and masses are generated. In the case of heterozygosity, two products with distinguishable mass are generated. Following PCR amplification, shrimp alkaline phosphatase (SAP) is added to dephosphorylate remaining dNTPs from the PCR cocktail. The samples are diluted with water and cation exchange beads (NH+ form) are added and the supernatant is used for MALDI-TOF MS analysis. A schematic representation of this reaction is presented in Fig. 1. Other approaches for MALDI-TOF-

based genotyping include the so-called invader[3- and

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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