Nucleic Acid Samples

The molecular sensitivities of the first and second generations of the bDNA assays were limited by nonspecific hybridization between the amplification probes and other nucleic acids. Short regions of hybridization between any of the probes constituting the amplification system, (preamplifier, amplifier, and labeled probe) and any nontarget nucleic acid sequence leads to amplification of the background signal. Capture probes, capture extenders, and sample nucleic acid are all sources of this background hybridization (Collins et al 1997). 

Masuda N, Ohnishi T, Kawamoto S, et al. Analysis of chemical modification of RNA from formalin-fixed samples and optimization of molecular biology applications for such samples. Nucleic Acids Res. 1999 27 4436 1443. 

Wang G, Brennan C, Rook M, et al. Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples. Nucleic Acids Res. 2004 32 (e76) 1-10. 

We have developed a simple method of nonisotopically labeling sample nucleic acids, which are then hybridized simultaneously to an array of unlabeled, immobilized probes. This "reversed hybridization" procedure thus provides identification results after a single hybridization reaction. 

A better method for highly parallel genetic analysis is needed. One with single molecule sensitivity that eliminates both the requirement for target amplification and the need for a target to be chemically modified or labeled for its detection would be ideal. Innovations in array construction, sample nucleic acid preparation... 

Northern blotting is analogous to Southern blotting except that the sample nucleic acid that is separated by gel electrophoresis is RNA rather than DNA. 

Finally, we wish to stress the dynamic nature of systematics and the necessary changes in nomenclature that accompany the discovery of new species or the reworking of evolutionary relationships within previously described taxa. Some molecular evolutionists may be naive about the importance of voucher specimens and the necessity of retaining information about the actual animals from which they have sampled nucleic acids or proteins. Voucher specimens physically and permanently document data in an archival report by (1) verifying the identity of the oiganism(s) used in the study and (2) by so doing, ensure that a study which otherwise could not be repeated can be accurately reviewed or reassessed. 9... 

Sample Nucleic Acid Protein Nitrogen Carbo- hydrate Crude Lipid... 

Maskos U, Southern EM. A novel method for the parallel analysis of multiple mutations in multiple samples. Nucleic Acids Res. 1993 21 2269-2270. 

Smith AM et al (2010) Highly-multiplexed barcode sequencing an efficient method for parallel analysis of pooled samples. Nucleic Acids Res 38 el42... 

Abstract. A model of the conformational transitions of the nucleic acid molecule during the water adsorption-desorption cycle is proposed. The nucleic acid-water system is considered as an open system. The model describes the transitions between three main conformations of wet nucleic acid samples A-, B- and unordered forms. The analysis of kinetic equations shows the non-trivial bifurcation behaviour of the system which leads to the multistability. This fact allows one to explain the hysteresis phenomena observed experimentally in the nucleic acid-water system. The problem of self-organization in the nucleic acid-water system is of great importance for revealing physical mechanisms of the functioning of nucleic acids and for many specific practical fields. 

The NAs such as DNA usually used in the experiments consist of 10" -1 o nucleotides. Thus, they should be considered as macrosystems. Moreover, in experiments with wet NA samples macroscopic quantities are measured, so averaging should also be performed over all nucleic acid molecules in the sample. These facts justify the usage of the macroscopic equations like (3) in our case and require the probabilities of finding macromolecular units in the certain conformational state as variables of the model. 

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... 

Nucleic acid (deoxyribonucleic acid (DNA) and ribonucleic acid (RNA)) probes utilize labeled, ie, radioactive, enzymatic, or fluorescent, fragments of DNA or RNA (the probe) to detect complimentary DNA or RNA sequences in a sample. Because the probe is tailored for one specific nucleic acid, these assays are highly specific and very sensitive (45). 

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. 

Factors to be considered in maldng the selection of chromatography processing steps are cost, sample volume, protein concentration and sample viscosity, degree of purity of protein product, presence of nucleic acids, pyrogens, and proteolytic enzymes. Ease with which different types of adsorbents can be washed free from adsorbed contaminants and denatured proteins must also be considered. 

In general, TAD shows better convergence than Cartesian dynamics. For nucleic acid strachires, for example, the convergence rate can be very low both for MMDG and for Cartesian dynamics owing to the low restraint density. The sampling of confomiational... 

A microscopic, ordered array of nucleic acids, proteins, small molecules, cells or other substances that enables parallel analysis of complex biochemical samples. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

A typical procedure is shown in Figure 2. Other dyes besides ethidium can be used, although ethidium has an advantage in that its excitation emission bands are well removed from any protein absorbances. A standard curve can be constructed for the nucleic acid of concern and the limits of detection established. In Step 3, proteolytic enzymes may be substituted for heparin, or the step may be bypassed in the case of proteins which do not interfere. After measurement of the unknown sample the nucleic acid concentration may be simply calculated or read from the standard curve. 

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