Base sequence determination

For most of the history of mankind, unraveling the nucleotide sequence of even a quite small nucleic acid was a formidable undertaking. Following 7 years of labor, Robert Holley solved the first such structure, that for an alanine tRNA from yeast, in 1961. This molecule contains a linear chain of 76 nucleotides and includes some unusual bases, which actually help in base sequence determination. For this achievement, Holley shared the Nobel Prize in Physiology or Medicine in 1968. 

At each stage in the peptide synthesis a second parallel synthesis is carried out on the same bead to attach the oligonucleotide tag (Figure 6.11). In other words, two alternating parallel syntheses are carried out at the same time. On completion of the peptide synthesis, the oligonucleotide tag is isolated from the bead and its base sequence determined and decoded to give the sequence of amino acid residues in the peptide. 

The principles of attachment of molecules to polysaccharides with concomitant insolubilization, discussed in the preceding two Sections, also apply to nucleic acids. The insolubilization of nucleic acids and polynucleotides provides materials useful (a) for fractionation and purification of other nucleic acids and related compounds,(b) for multiplication and isolation of single nucleic acid strands by base-pairing, " (c) for base-sequence determination, (d) as afiR-nants, " templates, and substrates for nucleic acid-related enzymes, and (e) as aflBnants for nucleic acid-binding proteins. ... 

Mass spectrometry sequencing of oligonucleotides is not as common a practice as it is for proteins and peptides. However, the following mass spectrometry-based sequence determination strategies have gained acceptance ... 

The anti-native DNA antibodies react equally well with DNAs which differ in their composition (DNA from mammalian cells, bacteria or virus) and thus it is unlikely that base sequence determinants are recognized, and specificity is essentially conformation-dependent. Antibodies reacting with native DNA have been described in several other human diseases (Koffler et al., 1969 Monier et al., 1970). However, the level was very low and their specificity has not been studied. 

Cambridge) the determination of base sequences in nucleic acids. 

The promise of being able to predict and modify the genetic characteristics of an organism fuelled massive efforts to determine the base sequences of human genes. The human genome project has now reached the goal of sequencing all the important DNA carried by humans. 

The stabilization of the radical cation by forming a polaron is a trade-off between its delocalization and the energy required to distort the DNA structure. The former lowers the kinetic energy of the intrinsically quantum mechanical migrating radical cation, and the latter will be determined by factors that are independent of specific base sequence, such as the force constants of bonds in the sugar diphosphate backbone. 

Fiskin, A. M., and M. Beer. 1965. Determination of Base Sequence in Nucleic Acids with the Electron Microscope. IV. Nucleoside Complexes with Certain Metal Ions. Biochem. 4,1287. 

Based on the results in this paper, the following experimental data should be obtained for each of the diastereoisomers. (l) The relative yields of trans and cis addition products should be determined for adduct formation to each base atom. (2) Alternating and non-alternating homopolymers should be used to evaluate the base sequence specificity. (3) Binding to sites IQ and IIX should be correlated to trans and cis adducts and to stereoselectivity. 

Edman Degradation. This technique requires more material than MS-based sequencing and its sensitivity decreases with the number of amino acids detected. The use of Edman degradation sometimes allows determination of those N-terminal amino acids that were not detected during MS sequencing. 

The mechanism of strand-directed mismatch correction has been demonstrated in E. coli (see, e.g., Wagner and Meselson, 1976). In this organism, adenine methyla-tion of d(G-A-T-C) sequences determines the strand on which repair occurs. Thus, parental DNA is fully methylated, while newly synthesized DNA is undermethylated, for a period sufficient for mismatch correction. By this means the organism preserves the presumed correct sequence, i.e., that present on the original DNA strand, and removes the aberrant base on the newly synthesized strand. Adenine methylation is achieved in E. coli by the dam methylase, which is dependent on S-adenosylmethionine. Mutants (dam) lacking this methylase are hypermutable, as would be expected by this model (Marinus and Morris, 1974). 

The successful expression of recombinant plant peroxidases such as HRP C has been a major focus of research in a number of laboratories. Three synthetic HRP C genes based on the amino acid sequence determined by Welinder (36, 47) were synthesized independently in order to initiate this work (63-65). A number of different expression systems have been evaluated (64, 66-73), a summary of which is presented in Table I. Refolding of recombinant HRP C isolated from inclusion... 

More recently, high-resolution chromatographic techniques (particularly reverse-phase and ion exchanged-based HPLC) have been applied in the separation of proteome proteins and high-resolution mass spectrometry is being employed to aid high-throughput sequence determination. 

The four bases whose sequence determines the hereditary information in ribonucleic acid (RNA), the naturally occurring polynucleotide, are adenine, cytosine, guanine, and uracil. These structures are as shown in Figure A. 

Direct recognition The order of bases can determine the pattern of weak interaction and the specificity of the complex formation. In this case there is a direct recognition of the sequence by the protein. 

An array of oligonucleotide which is composed of 20 80-mer oligos, or peptide nucleic acid probes, is synthesized either in situ (i.e., on-chip) or using conventional synthesis followed by on-chip immobilization. The resultant DNA array is then exposed to the labeled sample of DNA, hybridized, and the identity/abundance of complementary sequences determined. This method was developed at Affymetrix, Inc. and called DNA chips. Today, oligonucleotide-based chips are manufactured by many companies using alternative in situ synthesis or depositioning technologies. 

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