(a) Alignment
of Vsr homologs (2–7) from Arthrobacter luteus (Alu), Nocardia aerocolonigenes (Nae; U09581), E.coli (Eco; D90835), Xanthomonas oryzae (Xor; U06424), Bacillus subtilis (Bsu) and Haemophilus parainfluenzae (Hpa;
L17342). The region chosen for definition of degenerated primer
mixes is boxed. Where available, GenBank accession numbers for the
nucleotide sequences are given in parentheses. (b)
Sets of primer sequences coding for the amino acids D/H,
G/S, C, F, W and the first two bases of the H codon (W:
A or T; Y: C or T). The total number of different sequences within
a given primer set is indicated. (c) Cassette construct
consisting of two oligonucleotides with a central non-pairing bubble.
The cassette has a sticky end for ligation to GATC overhangs and
an EcoRI restriction site. The cassette primer
is complementary to the upper strand and its 3′-end
covers the bubble region in a way that elongation is possible only after
a first round of DNA synthesis with the upper strand as template.