Nucleic Acid Analyzers

A problem that has proved particularly difficult is the simplification of the sample preparation step for nucleic acid analyzes. Preparation of samples for DNA or RNA analysis is laborious. The traditional approaches for preparing nucleic acids can be classified into two categories liquid-liquid extraction and liquid-solid extraction. 

The Lawrence Livermore National Laboratory (LLNL) has developed a handheld-type device named Handheld Advanced Nucleic Acid Analyzer (HANAA) (Fig. 3) which is capable of rapid detection of bioagents such as B. anthracis in the field. It uses the TaqMan-based PCR assay system. Due to its small footprint and low weight, it is ideal for field applications. Another development from them is the Nanowire Barcode System which speeds up detection of pathogens such as anthrax, smallpox, ricin, and botulinum. Antibodies of specific pathogens are attached to the nanowires which produce small, reliable, and sensitive detection systems. 

Lab-on-Chip Devices for Biodefense Applications, Fig. 3 The Handheld Advanced Nucleic Acid Analyzer from Lawrence Livermore National Laboratory... 

Handheld Advanced Nucleic Acid Analyzer from Lawrence Livermore National Laboratory... 

Both the ease of use of this method for characterization of proteins and nucleic acids, and the abiHty to analyze many samples simultaneously for comparative purposes, have led to the prevalence of this technique. The drawbacks of a polyacrylamide matrix is that acrylamide is a neurotoxin, the reagents must be combined extremely carefiiUy, and the gels are not as pHable as most agarose gels. 

In Section II we provide an overview of the current status of nucleic acid simulations, including studies on small oligonucleotides, DNA, RNA, and their complexes with proteins. This is followed a presentation of computational methods that are currently being applied for the study of nucleic acids. The final section of the chapter includes a number of practical considerations that may be useful in preparing, performing, and analyzing MD simulation based studies of nucleic acids. 

VMD is designed for the visualization and analysis of biological systems such as proteins, nucleic acids, and lipid bilayer assemblies. It may be used to view more general molecules, as VMD can read several different structural file formats and display the contained structure. VMD provides a wide variety of methods for rendering and coloring a molecule. VMD can be used to animate and analyze the trajectory of a molecular dynamics (MD) simulation. 

The neutral hydrophilic surface and the wide range of pore diameters available for SynChropak GPC allow many compounds from small peptides to nucleic acids and other polymers to be analyzed. Table 10.2 lists the approximate exclusion limits for both linear and globular solutes. Although this information... 

Numerous organisms, both marine and terrestrial, produce protein toxins. These are typically relatively small, and rich in disulfide crosslinks. Since they are often difficult to crystallize, relatively few structures from this class of proteins are known. In the past five years two dimensional NMR methods have developed to the point where they can be used to determine the solution structures of small proteins and nucleic acids. We have analyzed the structures of toxins II and III of RadiarUhus paumotensis using this approach. We find that the dominant structure is )9-sheet, with the strands connected by loops of irregular structure. Most of the residues which have been determined to be important for toxicity are contained in one of the loops. The general methods used for structure analysis will be described, and the structures of the toxins RpII and RpIII will be discussed and compared with homologous toxins from other anemone species. 

Historically, the target analytes in clinical mass spectrometric applications were small, volatile compounds that could be analyzed by GC-MS (see Chapter 4). With time, new chemical preparation techniques and derivatization schemes broadened the scope of these metabolites to include fatty acids, amino acids, intermediates of glucose oxidation, phospholipids, steroids, neurogenic amines, nucleic acids, etc. The molecular weights (molar masses) after derivatization were less than 1000 Da, a mass range easily within the limits of most conventional mass spectrometers. 

A variety of buffers is used in electrophoresis. The selected buffer must contain ions to carry the current. Other than current-carrying capacity, the most critical criterion for buffer selection is the stability of the sample to be analyzed. Many proteins are unstable in acidic pHs, so alkaline buffers are frequently employed. Tris-(hydroxymethyl)amino methane (TRIS or THAM), sodium acetate, and ethylenedi-aminetetraacetate (EDTA) are common solutes in buffers, with pHs between 7.9 and 8.9 typical. (Refer to Chapter 5 for a discussion of buffers.) These buffers also work well with nucleic acid fragments. In addition, phosphate buffers, e.g., 10 mMK3P04, are often used with nucleic acid fragments (1.0 mM = 0.0010 M). 

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

GPC utilizes nonpolar organic mobile phases, such as THF, trichlorobenzene, toluene, and chloroform, to analyze for organic polymers such as polystyrene. GFC utilizes mobile phases that are water-based solutions and is used to analyze for naturally occurring polymers, such as proteins and nucleic acids. 

Electrophoresis is normally run in aqueous media, hence the analytes must be soluble in water. Presently only three types of water-soluble dendrimers have been successfully analyzed using gel electrophoresis techniques. The list includes Starburst PAMAM dendrimers [21], nucleic acid dendrimers [21] and poly(lysine) dendrimers [23, 24] (see Figures 10.2, 10.4 and 10.6). However, in each case appropriate water solubilizing terminal groups are required (i.e. -NH2, -OH or C02H groups) for suitable electrophoretic analysis. 

In situ hybridization (ISH) consists of the application of hybridization techniques to intact cells which demonstrate genetic information within a morphologic context. This technology takes advantage of the hybridization properties of nucleic acids and offers a distinct technique to directly analyze sequence information in intact tissues. In essence, it combines cytogenetic techniques with molecular biology to probe gene alterations at molecular levels. Development of... 

The Polymerase Chain Reaction. In the past, a major drawback of hybridization assays was their need for relatively large amounts of sample DNA to compensate for their low sensitivity. This problem has been surmounted in recent years by the development of powerful enzymatic techniques that can exponentially replicate specific DNA sequences in the test tube. With these techniques it is now possible to analyze vanishingly small samples that initially contain fewer than 10 copies of the sequence of interest. The new methods take advantage of the chemical properties of nucleic acids and of highly specialized enzymes that can repair and replicate DNA in vitro. 

Since 1974, evidence has accumulated in the literature which indicates that chromatin itself may be considered as an assembly system. It is true that chromatin is more complex than assembly systems analyzed to date, both with respect to the size of the nucleic acid involved and therefore the amount (and variety) of protein complexed with it and with respect to the dynamic aspect of the multilevel higher order structure. Nevertheless, at least at the lower levels of organization, the interpretation of chromatin as an assembly system may be valid. Evidence for this derives from three basic lines of research described in previous sections (1) the reconstitution of the nucleosome, (2) the self-assembly of the octamer, and (3) the putative self-organization of nucleosomes into higher order structures. 

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