A systematic DNA sequencing strategy

The final point to note is that sequencing by the primed synthesis methods will not reveal the presence of methylated or other modified nucleotides in the original DNA sequence. This is also true of the Maxam-Gilbert method where the DNA to be sequenced has been cloned into a plasmid vector. 

Recent experience in this laboratory has shown that such problems can be largely overcome by carrying out the elongation reaction and subsequent chase at 29°C rather than at room-temperature as previously described. 

In one experiment (Hindley, J., unpublished data) a set of dideoxy-nucleotide chain extension reactions, using an identical primed-template, was carried out at 14, 20 and 29°C respectively and the reaction products analysed on a standard thin sequencing 

Direct determination of sequences cloned into plasmid pBR322 

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