Results: Figure 3

In order to solve the problems that arise with longer primers, a slightly altered method was attempted, called single-site ribosubstitution.

Single-site ribosubstitution methods and concepts:

The methods are fairly similar to the previously described sequencing methods, however a single ribonucleotode rCTP, or ribo-CTP rather than a deoxy-ribo-CTP, is added to the incubation mixture and Mn buffer with mercaptoethanol is used rather than just H-buffer. Extension of the primer is carried out with the separate inhibitors, and the primer is split off the ribonucleotide by ribonuclease or alkali.

The method was done using both araCTP and ddCTP to illustrate that either inhibitor is useful when using this method.

110 nucleotides can be read in this sequence, however there are some difficulties. Between 3543-3550 there is some variation in the distance, perhaps indicating the formation of a loop. When the electrophoresis was done at a higher temperature the sequences reads GCTCGCG. Additionally some revision is required as the sequence before 3524-3530 should be TCAAC rather than ATTC-AC.

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