DNA sequencing methods

Genotypic resistance assays use DNA sequencing methods to examine the reverse transcriptase and protease regions of fhe HIV genome for all resistance-associated mutations. A major drawback of fhis fesfing mefhod is that results are difficult to interpret and expert consultation is necessary. 

Sanger and Barrell developed rapid DNA-sequencing methods. 

DNA polymerases (like RNA polymerases) are unique among enzymes in that the choice of substrate is determined by the template. E. coli polymerase can copy eukaryotic DNA and animal polymerases can copy bacterial DNA sequences given the appropriate DNA template. This means that DNA sequences, irrespective of their origin, can be accurately copied using E. coli DNA Pol I and this is the basis of the primed-synthesis DNA sequencing methods. 

Walter Gilbert (1932- ) was born in Boston, Massachusetts, and received a Ph.D. in mathematics at Cambridge University in 1957. He then joined the faculty at Harvard University as professor of physics, but bis research interests soon shifted to biochemistry. While still carrying out his academic work at Harvard, he founded Biogen, one of the earliest commercial biotechnology companies. He received the 1980 Nobel Prize in chemistry for his development of DNA sequencing methods. 

The dideoxy DNA sequencing method begins with the denaturation of double-stranded DNA (dsDNA) into single-stranded DNA. The ssDNA is then annealed with a fluorescent dye-labeled primer, which is an oligodeoxynucleotide 20 bases long. The heteroduplex formed is then incubated in four separate reactions. 

The above described PCR can only amplify the number of copies of a DNA molecules in the sample. In order to know what DNA or specific DNA sequence it is, however, one has to perform additional analyses, such as using gel electrophoresis or DNA sequencing method to find the answer. A direct DNA identification method, called the real-time PCR, combining both the PCR with fluorescent detection, is most useful in this regard. 

The discovery of site-specific restriction endonucleases in 1970 and the development of efficient DNA sequencing methods in 1977 sparked a revolution in biology. On Oct 1,1990, the 15-year project to sequence the complete human genome was officially begun. ... 

Rapid DNA-sequencing methods have allowed to determine the sequences of several membrane proteins. The mean hydrophobicity of 40 A long amino acid sequences in helical proteins with respect to a membraneous environment can thus be calculated. Most hydrophobic segments just correspond to the thickness of a bilayer membrane. 

Swerdlow H, Zhang J Z, Chen D Y, et al. (1991). Three DNA sequencing methods using capillary gel electrophoresis and laser-induced fluorescence. Analyt. Chem. 63 2835-2841. 

Progress during the past decade has resulted in the maturation of the triester methodology of chemical synthesis, sophistication in recombinant DNA techniques, and rapid DNA sequencing methods. It seems safer to predict that during the 1980s the large-scale and economical synthesis of important proteins, such... 

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