General background to DNA sequencing

DNA Polymerase I (DNA Pol I) from E. coli is the best characterized of all the DNA polymerases and, as far as DNA sequencing is concerned, is by far the most widely used. The purified enzyme is a single polypeptide chain of molecular weight 109,000 daltons containing approximately 1000 amino acid residues. The turnover number is about 667 nucleotides polymerized per molecule of enzyme per min at 37°C. One atom of zinc per molecule of enzyme appears to be required for the activity of the enzyme. 

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. 

Free 3 -hydroxyl ends may occur naturally in double stranded DNA or as a result of deliberate nicking (i.e. single stranded breakage) of the molecule with endonucleases such as DNAase I (Figs. 1.2. and 1.3.). Such nicks are usually found to be randomly distributed along a duplex DNA. In a linear duplex the presence of nicks has little effect on the physicochemical properties of the molecule and for that reason they are often referred to as hidden breaks. To discover whether a linear duplex DNA is nicked or not 

Gapped duplexes. The nicks introduced into single strands can be broadened into gaps by the action of exonuclease III (Fig. 1.3c.) 

Primed single strands. A single stranded DNA can be converted into an efficient template for DNA synthesis by annealing a short 

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