chapter  29
2 Pages

Determination of Transcription Initiation Site

The principle of this technique lies in hybridization in solution between mRNA of the gene studied (from a combination of mRNAs from the tissue studied) and a fragment of the genomic DNA of this gene, comprising the beginning of the coding part and a portion of the promoter sequence. This DNA sequence is first obtained by enzymatic restriction using restriction sites that flank the junction between promoter and coding sequence. In the example given, this is Nco I and Spe I. It is by analysing the sequence of this cloned DNA that the appropriate pair of enzymes is determined. This isolated DNA fragment is labelled radioactively at its 5 -P end (see Profile 11). The hybrid obtained occurs between the mRNA and the coding part of the gene. The promoter sequences remain single stranded. The use of a specific nuclease of single-stranded regions (SI nuclease) allows the elimination of these sequences and just the double-stranded hybrid can be retained. The size of this hybrid is determined on a sequencing-type gel (see Profile 23) in order to identify its first nucleotide base, which represents the initiation site of the gene transcription studied.