Nucleotide sequencing efficiency

Combinatorial Hbraries are limited by the number of sequences that can be synthesized. For example, a Hbrary consisting of one molecule each of a 60-nucleotide sequence randomized at each position, would have a mass of >10 g, weU beyond the capacity for synthesis and manipulation. Thus, even if nucleotide addition is random at all the steps during synthesis of the oligonucleotide only a minority of the sequences can be present in the output from a laboratory-scale chemical DNA synthesis reaction. In analyzing these random but incomplete Hbraries, the protocol is efficient enough to allow selection of aptamers of lowest dissociation constants (K ) from the mixture after a small number of repetitive selection and amplification cycles. Once a smaller population of oligonucleotides is amplified, the aptamer sequences can be used as the basis for constmcting a less complex Hbrary for further selection. 

The cell must possess the machinery necessary to translate information accurately and efficiently from the nucleotide sequence of an mRNA into the sequence of amino acids of the corresponding specific protein. Clarification of our understanding of this process, which is termed translation, awaited deciphering of the genetic code. It was realized early that mRNA molecules themselves have no affinity for amino acids and, therefore, that the translation of the information in the mRNA nucleotide sequence into the amino acid sequence of a protein requires an intermediate adapter molecule. This adapter molecule must recognize a specific nucleotide sequence on the one hand as well as a specific amino acid on the other. With such an adapter molecule, the cell can direct a specific amino acid into the proper sequential position of a protein during its synthesis as dictated by the nucleotide sequence of the specific mRNA. In fact, the functional groups of the amino acids do not themselves actually come into contact with the mRNA template. 

The cloning efficiency of the present method is sufficient to obtain positive clones from a single culture set, although artificial alterations of nucleotide sequences originating from the PCR process are inevitable. 

For sequencing such proteins, a complementary experimental approach based on recombinant DNA technology is often more efficient. As will be discussed in Chapter 6. long stretches of DNA can be cloned and sequenced, and the nucleotide sequence directly reveals the amino acid sequence of the protein encoded by the gene (Figure 4.29). Recombinant DNA technology is producing a wealth of amino acid sequence information at a remarkable rate. 

The nucleotide sequences of the four species (5S, 5.8S, 18S, and 28S) of rRNA have been determined, and these show remarkable homology among the three kingdoms (archaebacteria, eubacteria, and eukaryotes). In the case of ribosomal proteins, the amino acid sequences of about 35 proteins out of a total of about 80 have been determined. However, the exact roles of various rRNAs and proteins and their interactions in determining the activity, efficiency, and accuracy of the ribosomes is not very well understood at present. The coordinated synthesis of rRNAs and proteins, and the assembly of ribosomes is a process whose complexity is only beginning to be unravelled (Wool, 1991). 

Only certain DNA fragments are taken up efficiently by competent Haemophilus cells, which implies that efficient uptake requires the presence of a specific nucleotide sequence on the incoming DNA. Such a fragment has been identified as undecamer base pairs in common 5 -AAGTGCGGTCA-3. Its synthesis has been accomplished and has been shown to be biologically active in transporting DNA in the Haemophilus cell. ... 

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