Nucleotide sequencing automation

The ability to manufacture oligos of specified nucleotide sequence is relatively straightforward using automated synthesizers. 

The individual components of the cDNA library may be obtained by grouping individual bacteria on a culture medium so that they reproduce to form identical clones. This enables a large quantity of specific cDNAs to be produced in a pure form. The cDNA within these plasmid-containing bacteria can then be removed, and the precise nucleotide sequence determined by standard automated analytical procedures. 

The recent revolution in molecular biology has brought with it an increased demand for the efficient chemical synthesis of short DNA segments, called oligonucleotides. The problems of DNA synthesis are similar to those of protein synthesis (Section 26.10) but are more difficult because of the complexity of the nucleotide monomers. Each nucleotide has multiple reactive sites that must be selectively protected and deprotected at the proper times, and coupling of the four nucleotides must be carried out in the proper sequence. Automated DNA synthesizers are now available, however, that allow the fast and reliable synthesis of DNA segments up to 200 nucleotides in length. 

Fig. 1. Amino acid sequence of GMF-beta. The sequence was established for bovine brain GMF-beta by automated Edman degradation (microsequencing) and tandem mass spectrometry. Identical sequence was obtained for recombinant hGMF-beta, which was deduced from nucleotide sequence of the cDNA and verified by microsequencing of the first ten NH2-terminal residues and by carboxylpeptidase de adation of the first four COOH-terminal residues. The one-letter abbreviations for the amino acids are A, Ala C, Cys D, Asp E, Glu F, Phe G, Gly H, His I, He K, Lys L, Leu M, Met N, Asn P, Pro Q, Gin R, Arg S, oer T, Thr V, Val W, Trp and Y, Tyr. The three cysteine residues (at positions 7, 86 and 95) are underlined. (Adapted from ref. 13)...

Fig. 4. Schematic diagram of the steps in the automated PCR/OLA procedure performed with a robotic workstation. The assay contains three steps (1) DNA target amplification (2) analysis of target nucleotide sequences with biotin (B)-labeled and digoxigenin (D)-labeled oligonucleotide probes and T4 DNA ligase (L) and (3) capture of the biotin-labeled probes on streptavidin (SA)-coated microtiter wells and analysis for covalently linked digoxigenin by using an ELISA procedure with alkaline phosphatase (AP)-conjugated antidigoxigenin (aD) antibodies and a substrate (S). Reprinted with the permission of Nickerson el al. (N2) and the Proc. Natl. Acad. Sci. (U.SA.).

Schaller H, Weiman G, Lerch B, Khorana HG. The stepwise synthesis of specific deoxypolynucleotides. J Am Chem Soc 85, 3821-3827, 1963. Usman N, Ogilvie KK, Jiang M-Y, Cedergren RJ. Automated chemical synthesis of long oligoribonucleotides using 2 -0-silylated libonucleoside 3 -0-phosphoramidites on a controUed-pore glass support Synthesis of a 43-nucleotide sequence similar to the 3 -half molecule of Escherichia coli formylmethionine tRNA. J Am Chem Soc 109 7845-7854, 1987. 

Facile experimental techniques to determine nucleotide sequences have resulted in several protein primary structures for the same molecular type but in different species for instance, at least 226 hemoglobin sequences are known for bacteria to man (31). This plethora of data has necessitated automated, multiple sequence alignment. The challenges are formidable and no general solution has yet been found. 

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