Nucleic acid sequencing components

Molecular biology involves the study of the major macromolecules, DNA, RNA, and protein. The central dogma ofmolecular biology is illustrated in Fig. 2. The central dogma shows the relationship among the macromolecules in the processes of transcription and translation. Figure 2 also gives the relationship between immunoelectron microscopy and in situ hybridization. In situ hybridization allows one to localize a specific nucleic acid sequence. Immunoelectron microscopy is an essential component to the technique of in situ hybridization when applied at the EM level. 

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. 

RNA has three basic roles in the cell. First, it serves as the intermediate in the flow of information from DNA to protein, the primary functional molecules of the cell. The DNA is copied, or transcribed, into messenger RNA (mRNA), and the mRNA is translated into protein. Second, RNA molecules serve as adaptors that translate the information in the nucleic acid sequence of mRNA into information designating the sequence of constituents that make up a protein. Finally, RNA molecules are important functional components of the molecular machinery, called ribosomes, that carries out the translation process. As will be discussed in Chapter 2, the unique position of RNA between the storage of genetic information in DNA and the functional expression of this information as protein as well as its potential to combine genetic and catalytic capabilities are indications that RNA played an important role in the evolution of life. 

An additional nucleic acid sequence, a probe, may be included in a reassociation reaction and form double strands with components of the initial DNA. This hybridization reaction is the basis of the Southern and northern blotting techniques used to determine if a known nucleic-acid sequence is present in a mixture of nucleic-acid sequences. 

The process is initiated with a random library of linear oligonucleotides (usually, 10 to 10 ) consisting of linear nucleic acids comprising a random sequence embraced by a 5 and a 3 nucleic acid sequence of defined composition. An RNA-searched aptamer involves the primary transcription of the DNA library into an RNA pool followed by passing the library through a separating matrix that includes the target substrate. The few nucleic acids that reveal affinity toward the substrate (or some nonspecific nucleic acid adsorbents) bind to the separation matrix, while most of the library components are washed off. The elution of surface-bound nucleic acids followed by their polymerase chain reaction (PCR) amplification yields a mixture of nucleic acids... 

Structure. The mononucleotides of RNA consist of ribose phosphorylated at C3, and linked by an N-gly-cosidic bond to one of four bases adenine, guanine, cytosine or uracil. Many other bases (chiefly methylated bases) also occur, but are less common (see Rare nucleic acid components). The mononucleotide units form a linear chain via 3, 5 phosphodiesler bonds (see Nucleic acids). Sequence analysis of RNA has become a standard technique. In many cases the amino acid sequences of proteins are predicted from the sequence of the corresponding mRNA (or DNA) because it is much easier to clone the nucleic acid than to isolate the protein. 

Tautz, D. and Renz, M. (1984) Simple sequences are ubiquitous repetitive components of eukaryotic genomes. Nucleic Acids Research 12, 4127-4138. 

---