Selective cloning of a defined number of tandem DNA repeats in Escherichia coli.

Abstract

Repetitive DNA sequences play an important role in biology, especially at the level of eucaryotic gene transcription control. Their construction, assembly and cloning by conventional means or by PCR derived methods is usually very cumbersome. This method is based on the association of a small DNA tag with the sequence to be polymerised. The multimeric sequence is constructed in a stepwise manner, the tag allowing for simple in vivo selection of the (n+1) polymer containing vector at each step. Thus, n rounds of cloning generate a family of vectors, each harbouring a defined number of ordered tandem copies of the sequence of interest, ranging from 1 to n. Contrary to the multimerization methods based on PCR, this method is independent of the size of the sequence to be polymerised. It is, in fact, especially well suited for the moderate polymerisation of short DNA sequences such as regulatory proteins binding sites, or the preparation of small size DNA ladders. The outline of the method is as follows (Fig. 1). The sequence to be polymerised is first cloned between two compatible ends restriction sites in a cloning vector (sites A and B), generating vector 1X. Then, a small cassette encompassing an amber suppressor tRNA gene with its promoter and transcription terminator is cloned next to it (between sites C and D), to obtain vector 1X+S which is used to transform an amber mutated strain, such as, for example, XAC-1 (1). The 2X+S vector is obtained by cutting out the 1X+S fragment with restriction enzymes A and D, and cloning it between sites B and D of vector 1X. Growth of the transformants on minimal medium ensures that only the correct construct is selected for. In the same manner, the 3X+S vector is obtained by cloning the 2X+S A/D fragment into the 1X vector between the B and the D sites and selecting the transformants for the presence of the suppressor gene. The 3X+S vector will thus contain three adjacent copies of the sequence of interest, in the same orientation, between two unique restriction sites A and B, as the sites between the copies are destroyed by the cloning procedure and the asymmetrical cloning ensures that only one orientation of the sequence is inserted. Each time the cloning step is repeated, one unit of the sequence is added to the polymerised sequence. With a recombination deficient amber mutated strain as a host for the successive transformations, the limit to the number of copies of the sequence of interest is only given by the size of the plasmid. If the nX+S containing vector is destroyed by additional restriction cuts when preparing the nX+S fragment, then no purification of this fragment is required before cloning, and the successive rounds of cloning are very simple. Figure 1. Outline of the method. A and B are compatible ends restriction sites such as SalI and XhoI for example.