| Spelling out the answer. In
the much-automated Sanger sequencing method, the
single-stranded DNA to be sequenced is "primed"
for replication with a short complementary strand at one
end. This preparation is then divided into four batches,
and each is treated with a different replication-halting
nucleotide (depicted here with a diamond shape), together
with the four "usual" nucleotides. Each
replication reaction then proceeds until a
reaction-terminating nucleotide is incorporated into the
growing strand, whereupon replication stops. Thus, the
"C" reaction produces new strands that
terminate at positions corresponding to the G's in the
strand being sequenced. (Note that when long strands are
being sequenced the concentration of the
reaction-terminating nucleotide must be carefully chosen,
so that a "normal" C is usually paired with a
G; otherwise, replication would typically stop with the
first or second G.) Gel electrophoresis -- one lane per
reaction mixture -- is then used to separate the
replication products, from which the sequence of the
original single strand can be inferred. |
