End-labelling DNA segments

The starting material for sequence analysis is usually a defined duplex DNA fragment purified from a restriction enzyme digest or obtained by a cleavage, at a unique site, of a defined circular DNA from a plasmid, phage or virus. Unique ends may also originate in 

3 -end labelling can be carried out using two different reaction schemes. In the presence of calf thymus terminal transferase and a ribonucleoside triphosphate a variable number of ribonucleotide residues are polymerized onto the 3 -ends of DNA strands in a template-independent reaction. If the triphosphate is labelled with [32P] in the alpha position, they will label the 3 -ends of the DNA. 

Maxam and Gilbert (1980) have described detailed protocols for the preparation and isolation of dephosphorylated DNA fragments, and reaction schemes for the selective 5 -end and 3 -end labelling of these products either individually or en masse. Pro- 

Procedure A describes the preparative scale isolation of DNA restriction fragments and their dephosphorylation with alkaline phosphatase. 

Procedure B describes methods for the 5 -end labelling of dephos-phorylated fragments with either protruding 5 -ends or with flush or recessed 5 -ends. 

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In the dimethylsulfate (DMS) technique, the end-labeled DNA segment is treated with DMS, cleaved chemically at its methylated G residues, and the resulting fragments separated electrophoretically this entire procedure is described in detail in the entry Nucleic acid sequencing (see). As in the previous technique, gaps or footprints in the autoradiogram identify the position and nature of the DNA sequences that bind the protein in question. 

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