Nucleic acids sequence determination

This chapter has several purposes to provide an overview of existing nucleic acid sequencing approaches, to indicate areas of rapid technical development, and to serve as an introduction to nucleic acid sequence determination. Also, it may provide a slightly different perspective for those already familiar with sequencing routines. Thus, this chapter is not intended to serve as a source of all available techniques. 

As already noted, templated sequencing protocols are now the most generally used methods for nucleic acid sequence determination. The use of the highly processive modified bacteriophage T7 polymerase (Sequen-ase U.S. Biochemical Corp., Cleveland, OH) has eliminated many earlier problems associated with variable intensity of individual bands on sequencing gels. 

This chapter describes the chemistry of nucleotides and the m or classes of nucleic acids. Chapter 12 presents methods for determination of nucleic acid primary structure (nucleic acid sequencing) and describes the higher orders of nucleic acid structure. Chapter 13 introduces the molecular biology of recombinant DNA the construction and uses of novel DNA molecules assembled by combining segments from other DNA molecules. 

This assay system developed by Chaires [136] is a new, powerful and effective tool based on the fundamental thermodynamic principle of equilibrium dialysis for the discovery of ligands that bind to nucleic acids with structural and sequence selectivity. Here, identical concentrations of various nucleic acid samples are dialysed in dispodialysers against a common ligand solution. At equilibrium, the contents of the ligand bound to each nucleic acid are determined and this is correlated directly to the ligand s specificity to a particular sequence. 

The viral load test quantifies viremia by measuring the amount of viral RNA. There are several methods used for determining the amount of HIV RNA reverse transcriptase-coupled polymerase chain reaction, branched DNA, and nucleic acid sequence-based assay. Each assay has its own lower limit of sensitivity, and results can vary from one assay method to the other therefore, it is recommended that the same assay method be used consistently within patients. 

The last section of this chapter includes in brief a procedure of McF adden (9) for in situ hybridization. In situ hybridization relies on the complementarity of the bases contained within DNA and RNA. In addition to the hybridization (reassociation) of complementary DNA strands, hybridization is possible between DNA and RNA strands that are complementary. Also, hybridization is possible between a synthetic sequence and a sequence of biological origin. In situ hybridization may be used to determine the location of a specific nucleic acid sequence within a cell. The procedure requires the use of a probe for the sequence of interest. The probe, in turn, must be complementary to the sequence of interest. The probe may be either single stranded or double stranded, DNA or RNA. There must exist a method by which to detect the probe. 

Determined by protein or nucleic acid sequence analysis. ND, not determined. 

Currently, there is a need for high-throughput determination of nucleic acid sequences. At present, detection systems most commonly employ fluorescence-based methods. However, wide spread applications of such methods are limited by low speed, high cost, size, and number of incubations steps, among other factors. Application of electrochemical methods in affinity DNA sensors presents likely a promising alternative, allowing miniaturization and cost reduction, and potentially allowing application in point-of-care assays. 

Selective capturing of the active nucleic acid sequences is a crucial factor in determining the success of an RNA catalyst in vitro selection. A convenient technique for partitioning active from inactive RNA sequences involves capture of biotin by streptaviclin. Biotin is a widely used affinity tag because of its strong binding (K 10-15) to streptavidin. Potential substrates are typically synthesized with... 

Restriction enzymes allow DNA to be cut at specific sites nucleic acid hybridization allows the detection of specific nucleic acid sequences DNA sequencing can be used to easily determine the nucleotide sequence of a DNA molecule. 

Human apoA-I is a major constituent of HDL, with an Mr of approximately 28,300, calculated from the known primary structure (Bl, B43). ApoA-I is initially synthesized as a 267-amino-acid precursor protein, pre-pro-apoA-I (G25, G26), containing an 18-amino-acid prepeptide and a 6-amino-acid propeptide [determined by nucleic acid sequence analysis of cloned apoA-I (L6), and by isolating the primary translation product of human intestinal apoA-I mRNA (G25)]. 

Many ancillary formulations have been proposed for analytical purposes, which frequently include an ascorbate as antioxidant. Although this component has no direct function in the analytical process, it is claimed to improve the performance of the method. This is the case of sodium ascorbate added to rat bile samples while determining tetrahydrofolate or while determining urinary copper ion concentration. This is also the case of ascorbate added to buffers used in nucleic acid sequencing based on FLD. The titrimetric determination of Mn(III) or Ni(III) in presence of excess of ascorbic acid ° and the GC-ECD determination of trihalomethane in water are based on a similar approach. 

The identity of the positive library compound that interacts with the target protein can be determined by knowing the nucleic acid sequences of the oligonucleotide microarrays. 

Error = 100 x (experimental value - trae value) / (true value) Average % Error = (Z1% Error for 16 amino acidsi) /16 These calculations were repeated substituting the true value with analyzed value . Analyzed values are the overall average determined by all the participants and may represent a more realistic figure than the theoretical values, which were determined by protein or nucleic acid sequencing. These values were used to compare the errors of the pre-hydrolyzed and protein samples. In analyzing data, we excluded sites with errors >3 standard deviations from the mean. 

Nucleic acid sequences were presented in almost every chapter in this textbook-Determination of these sequences requires a DNA sequencing technique. The... 

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