Genomic quantification

Genomic quantification is necessary to identify large deletions or duplications that occasionally cause single gene disorders. A novel molecular method (multiplex ligation-dependent probe amplification, MLPA) has been recently developed for this purpose and is presented in this chapter. 

Fig. 8.2.2 Principle of genomic quantification by multiplex ligation-dependent probe amplification (MLPA). Synthetic oligonuleotides are designed to bind to exon 1 (oligo 1a and 1b) and 2 (2a and 2b). After specific hybridisation, the oligos are ligated. The ligation products undergo quantitative polymerase chain reaction using universal primers X and Y...

A prototype bDNA assay was developed for quantification of HGV/GBV-C RNA in serum (Pessoa et al, 1997). The assay employed target probes based on the relatively conserved sequence in the 5 untranslated region of the HGV/GB V-C genome. Preamplifier molecules and incorporation of isoC and isoG into the sequences common to bDNA assays were used to enhance the analytical sensitivity. The provisional limit of detection was 32,500 genome equivalents/ml based on dilutions of a 700-nucleotide synthetic HGV/GBV-C RNA transcript. The run-to-run variance of the assay was <15%. 

Fluorescently labeled DNA probes can be used for detection, localization, or quantification of target DNA sequences. In situ hybridization mapping of genomic DNA sequences can be... 

It is clear that in this brief overview of molecular biology, we have not covered a number of important areas that have an important impact on the study of metalloproteins. These include molecular cloning and recombinant DNA technology, which allow proteins to be over-expressed and individual amino acids to be mutated to any other of the 19 protein amino acids genome and proteome analysis that enables the sequences of all the genes of the entire organisms to be determined, and the quantification, localization, interactions and, where possible, activities and identification of all of the proteins in an organism,... 

Hayward-Lester A, Chilton BS, Underhil PA, Oefher PJ, Doris PA. 1997. Quantitation of spedfic nudeic adds, regulated RNA processing and genomic polymorphism using reversed-phase HPLC. Gene quantification. Ferre F, editor. Boston Birkhauser. 

Proteomics is concerned with the analysis of the complete protein complements of genomes. Thus proteomics includes not only the identification and quantification of proteins, but also the determination of their localization, modifications, interactions, activities, and functions. This chapter focuses on protein sequences as the sources of biochemical information. Protein sequence databases are surveyed. Similarity search and sequence alignments using the Internet resources are described. 

Proteome refers to protein complement expressed by a genome. Thus proteomics concerns with the analysis of complete complements of proteins. It is the study of proteins that are encoded by the genes of a cell or an organism. Such study includes determination of protein expression, identification and quantification of proteins as well as characterization of protein structures, functions and interactions. The functional classification of proteins in genomes (i.e., proteomes) can be accessed from the Proteome Analysis Database at http //ebi.ac.uk/proteome/ (Apweiler et ah, 2001). 

Since alterations in global DNA methylation are implicated in various pathobio-logical processes, a gradient IPC-ESI-MS/MS method with a volatile IPR was used to determine cytosine and 5-methylcytosine in DNA quantification relied on stable isotope dilution [58], Muscular dystrophies caused by various mutations in the dystrophin gene are amenable to easier prenatal diagnosis via a multiplex polymerase chain reaction (PCR)/IPC assay [59]. Some guidelines for the analysis of genomic DNA by IPC-ESl-MS can be found in Reference 60. 

The dimensionality of chemical structure space exceeds that of known biological functional space. The dimensionality of biological functional space has increased in recent years due to the discovery of a multitude of genes, largely from the Human Genome Project. This chapter, however, will focus on chemical diversity rather than functional diversity. Quantification of chemical diversity involves two areas first, the predefmition of a chemical space, accomplished by selection of a diversity metric and a compound representation (i.e., molecular descriptors) and second, a rational subset selection, or classification, method dependent on efficient dimensionality reduction. Here, we describe these methods, prerequisites for a definition... 

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