A degenerate primer (PCT4) was designed with the sequence
KKVRVRV(CT)6, where K = G/T, V = G/C/A and R = G/A. The
(CT)6 component of the primer was designed to anneal to (GA)n,
where n . 6, and the seven 54 nucleotides in the primer form the
‘anchor’. The two most 54 nucleotides in the anchor were designed
to anneal to any nucleotide, with G used to pair with C or T, and
T used to pair with A or G. The next five nucleotides, designated
‘blocking’ bases, were designed so that they would not pair with
GA repeats, but were otherwise as redundant as possible. Thus V
(G, A or C) will pair with any nucleotide except A, while R (G or
A) will not pair with G. Also, R will not pair with A because T was
excluded from the base to avoid T-G binding. These redundancies
mean that this primer should be complementary to one in six of all
possible random sequences adjacent to GA repeats.