Electrophoresis DNA sequencing

Kheterpal I. and Mathies R. A., Capillary array electrophoresis. DNA sequencing, Anal. Chem. News Features, 71 (1), 31A, 1999. 

Kheterpal I, Mathies RA. Capillary array electrophoresis DNA sequencing. Analyt. Chem. 1999 71 31A-37A. 

There are several forms of electrophoresis. In slab gel electrophoresis the conducting buffer is retained within a porous gel of agarose or polyacrylamide. Slabs are formed by pouring the gel between two glass plates separated by spacers. Typical thicknesses are 0.25-1 mm. Gel electrophoresis is an important technique in biochemistry, in which it is frequently used for DNA sequencing. Although it is a powerful tool for the qualitative analysis of complex mixtures, it is less useful for quantitative work. 

A potentially general method of identifying a probe is, first, to purify a protein of interest by chromatography (qv) or electrophoresis. Then a partial amino acid sequence of the protein is deterrnined chemically (see Amino acids). The amino acid sequence is used to predict likely short DNA sequences which direct the synthesis of the protein sequence. Because the genetic code uses redundant codons to direct the synthesis of some amino acids, the predicted probe is unlikely to be unique. The least redundant sequence of 25—30 nucleotides is synthesized chemically as a mixture. The mixed probe is used to screen the Hbrary and the identified clones further screened, either with another probe reverse-translated from the known amino acid sequence or by directly sequencing the clones. Whereas not all recombinant clones encode the protein of interest, reiterative screening allows identification of the correct DNA recombinant. 

Eor example, the technique of Southern blotting was developed (68) for use with agarose gel electrophoresis of DNA fragments. Southern blots are designed to detect specific sequences of DNA. After electrophoresis is complete, the DNA is denatured and the single stranded DNA transferred to the specially prepared nitrocellulose paper. The nitrocellulose is then incubated with radioactive RNA or DNA complementary to those DNA sequences of interest. After the nitrocellulose has been sufftciendy incubated with the radioactive complementary DNA, autoradiography is used to identify the fragments of interest. 

Southern, E. M., 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. Journal of Molecular Biology 98 503-517. The classic paper on die idendficadon of specific DNA sequences through hybridizadon widi unique probes. 

G. A. Kowalchuk, J. R. Stephen, W. DeBoer, J. 1. Prosser, T. M. Embley, and J. W. Woldendorp, Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified I6S ribosomal DNA fragments. Appl. Environ. Microbiol. 6.1 1489 (1997). 

Pentoney, Jr., S. L., Konrad, K. D., and Kaye, W., A single-fluor approach to DNA sequence determination using high performance capillary electrophoresis, Electrophoresis, 13, 467, 1992. 

Resolution in capillary gel electrophoresis of DNA sequencing was shown to be directly proportional to the product of the number of bases and the relative peak distance, i.e., to the mean separation of peaks.43 Reformulation of the treatment of the capacity factor has been used to simplify and clarify the interpretation of the separation factor in electrophoresis.44 Peak... 

Kim, Y. and Yeung, E.S., Separation of DNA sequencing fragments up to 1000 bases by using poly(ethylene oxide)-filled capillary electrophoresis, ]. Chro-matogr. A, 781, 315, 1997. 

Myers, R.M., Fischer, S.G., Maniatis, T. and Lerman, L.S. (1985b) Modification of the melting properties of duplex DNA by attachment of a GC rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Research 13, 3111-3129. 

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