Sequencing of nucleic acids

The amount of sample required is quite small as little as 10 mole is typical So many peptides and proteins have been sequenced now that it is impossible to give an accurate count What was Nobel Prize winning work m 1958 is routine today Nor has the story ended Sequencing of nucleic acids has advanced so dramatically that it is possible to clone the gene that codes for a particular protein sequence its DNA and deduce the structure of the protein from the nucleotide sequence of the DNA We 11 have more to say about DNA sequencing m the next chapter... 

It is only natural that, to date, bioinformatics tools contribute most to the analysis of amino acid sequences. Only a small amount of current sequence data is subjected to direct experimentation. The majority of amino acid sequences currently accessible in public databases have been derived by in silico translations of nucleic acid sequence data, despite the fact that amino acid sequencing was introduced historically long before nucleic acid sequencing. It is hard to predict the future of the experimental generation of primary data. Certainly, sequencing of nucleic acids continues to become cheaper and faster, and novel techniques may further enhance the production of data. DNA chips are already used to detect differences between very similar sequences other methods may generate DNA data even more efficiently. 

A selective method of preventing the expression of adhesion molecules or cytokines is the use of antisense oligonucleotides. These oligonucleotides are short sequences of nucleic acids complementary to mRNA sequences of specific proteins of interest. If delivered to the cytoplasmic compartment of cells these oligonucleotides are able to form a complex with their target mRNA. In this way the translation of mRNA into protein by ribosomes is inhibited. The subsequent mRNA degradation by RNAse H results in reduced expression of the protein (see also Chapter 5 for a description of antisense ohgonucleotides as therapeutic modalities). 

Cell components or metabolites capable of recognizing individual and specific molecules can be used as the sensory elements in molecular sensors [11]. The sensors may be enzymes, sequences of nucleic acids (RNA or DNA), antibodies, polysaccharides, or other reporter molecules. Antibodies, specific for a microorganism used in the biotreatment, can be coupled to fluorochromes to increase sensitivity of detection. Such antibodies are useful in monitoring the fate of bacteria released into the environment for the treatment of a polluted site. Fluorescent or enzyme-linked immunoassays have been derived and can be used for a variety of contaminants, including pesticides and chlorinated polycyclic hydrocarbons. Enzymes specific for pollutants and attached to matrices detecting interactions between enzyme and pollutant are used in online biosensors of water and gas biotreatment [20,21]. 

A useful approach to monitor microbial populations in the biotreatment of hazardous wastes involves the detection of specific sequences of nucleic acids by hybridization with complementary oligonucleotide probes. Radioactive labels, fluorescent labels, and other kinds of labels are attached to the probes to increase sensitivity and simplicity of the hybridization... 

The nucleotide sequences of nucleic acids can be represented schematically, as illustrated on the following page by a segment of DNA with five nucleotide units. The phosphate groups are symbolized by (3, and each deoxyribose is symbolized by a vertical line, from C-l at the top to C-5 at the bottom (but keep in mind that... 

Because nucleic acids contain a large number of nucleotides, biochemists have devised an abbreviation system to indicate the nucleotide sequence of nucleic acids (see Fig. V-2). This system assumes the 3, 5 -phosphodiester linkages and lists, in a 5 to 3 order, the nucleotide sequence of the nucleic acid with p (phosphate) between each nucleotide. Using this latter system, the RNA nucleotide sequence shown in Figure V-2 is. .. ApGpCpUp. You may also encounter an even simpler abbreviation system where the phosphate (p) between each nucleotide is omitted (e.g.,. . . AGCU. . . ). 

With a small adjustment to this procedure, using thiophosphoryl chloride (PSCI3) in place of phosphoryl chloride (POCI3), the nucleoside 5 -(l-thio)triphosphates can be easily synthesized.7 These compounds have been used extensively for applications inter alia for site-directed mutagenesis, sequencing of nucleic acids and investigation of enzyme mechanisms.16 Phosphorylation with thiophosphoryl chloride is generally slower than with phosphoryl chloride but still occurs at a reasonable rate for purine nucleosides. However, for pyrimidine nucleosides, it is necessary to add 2,4,6-collidine as a catalyst, which forms a reactive intermediate with the thiophosphoryl chloride in situ. 

Solid-phase synthesis of nucleic acids. Precise sequences of nucleic acids can be synthesized de novo and used to identify or amplify other nucleic acids. 

DNA probe— An agent that binds directly to a predefined sequence of nucleic acids. 

Complementary base sequence. For a given sequence of nucleic acids, the nucleic acids that are related to them by the rules of base pairing. 

H. Oberacher, B. Wellenzohn, C.G. Huber, Comparative sequencing of nucleic acids by LC-MS-MS, Anal. Chem., 74 (2002) 211. 

The sequencing of nucleic acids remains one of the most challenging applications of mass spectrometry. New technological developments centered around the ionization process have brought an unexpectedly quick answer to this problem. Although species with molecular masses of several million daltons can not yet be handled, the sequencing of shorter polynucleotides is easily done, using the technique of fast atom bombardment... 

FAB) coupled to a data system to simplify the task of analyzing the complex FAB spectra. This is the latest and certainly the most active research area in state-of-the-art mass spectrometry of nucleic acids. There is little doubt that this positive response to the challenge involved in the sequencing of nucleic acids should allow mass spectrometry to occupy a select position among modern analytical techniques. 

B. R. Kowalski, Anal. Chem. 47, 1152A (1975) also see his previous papers on similar computer applications not related to the sequence of nucleic acids problem. 

Alternatives to electrophoresis Determines the size or sequence of nucleic acid without use of electrophoresis. Examples are high-performance liquid chromatography (HPLC) and mass spectrometry. 

Figure 20. A diagram illu.< trating phylogenetic relationships between the three kingdoms of organisms. The data used to determine these come from the base sequences of nucleic acids. The tree presently has no root because u f have no unambiguous kmtwledge of the ancestral forms (from Engel and Macko, 1993).

Oberacher, H. Mayr, B.M. Huber, C.G. Automated De Novo Sequencing of Nucleic Acids by Liquid Chromatography-Tandem Mass Spectrometry, J. Am. Soc. Mass Spectrom. 15(1), 32 2 (2004). 

These techniques rely on the specificity provided by the hybridization between two complementary (or nearly so) sequences of nucleic acids. Sequences can be selected for probing that are very common among organisms, for functional or phylogenic groups, or very specific for only one strain of an organism. 

Mohanty U, Searls T, McLaughlin LW. Anomalous migration of short sequences of nucleic acids in polyacrylamide gels prediction and experiment. J Am Chem Soc 1998 120 8275-8276. 

The most important applications of electrophoresis are in molecular biology and medicine where, for example, the study of inherent variabilities of serum proteins has produced a new branch of genetics, and the discovery of hemoglobin variants in several anemias has introduced the notion of molecular diseases. Electrophoresis has also greatly facilitated sequencing of nucleic acids, the clinical diagnosis of protein dyscrasias, the measurement of isoenzyme distribution, and the classification of lipoproteinemias, among others. 

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