Diagnostic Applications

MALDI-TOF MS of nucleic acids after base-specific cleavage improves the odds of differential sequence analysis and sets a significant milestone in the field of comparative genomics and genetics. The feasibility of the system exceeds SNP discovery applications. The method facilitates efficient scoring of large numbers of genetic markers in selected populations determining genotypic and phenotypic correlations.

Base-specific cleavage of signature sequences results in species-specific mass signal pattern of the region of interest. These species-specific fingerprints can be utilized to discriminate prokaryotes—bacterial or viral organisms—to the genus-, species-, or strain-specific level and thus facilitate pathogen identification.1-15-1 Figure 3 gives an example of the unambiguous identification and differentiation of four mycobacterial species based on their characteristic mass signal fingerprints of the 16S rRNA gene sequences. All expected signals were identified in the spectra and unique identifier signals marked by arrows could be unambiguously assigned to expected discriminatory species-specific fragments (Fig. 3).

Further expansion of the application portfolio includes the detection of epigenetic modifications, molecular haplotyping, and mutation detection.

Analysis of DNA methylation-mediated gene silencing is seen as a valuable diagnostic tool in cancer research and diagnostics. Qualitative as well as quantitative high-throughput MALDI-TOF DNA methylation analysis relies on bisulfite treatment of genomic DNA, PCR amplification followed by base-specific cleavage. The bisulfite treatment converts nonmethylated cytosine to uracil, while methylated cytosines remain unmodified. As a result, methylated vs. nonmethylated mass signal patterns show significant differences, which allow for differentiation as well as identification of individual sites of methylation in a target sequence. In addition, comparative quantitation of mass signals can be utilized to determine the relative abundance of methylated vs. nonmethylated target gene regions.[16]

Haplotypes are defined as the collection of genotypes found in a single allele or chromosome. Their unambiguous identification provide additional power in the detection of genes involved in common diseases. This can contribute to a better understanding of the complex etiology of diseases. Long-range, allele-specific PCR using nonextendable exonuclease resistant competitor oligonu-cleotides is performed to isolate desired alleles/chromo-somes prior to base-specific cleavage and MALDI-TOF detection. The analysis is not exclusive to known markers, and novel associated subhaplotypes can be discovered resulting in comprehensive haplotype information of the loci of interest.[17]

Additional fields of application include large-scale characterization of cDNAs and alternative splice variants—further steps in the attempt to elucidate the genetic code and individual variations.

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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