|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.|
To Know Ourselveswas prepared at the request of the U.S. Department of Energy, Office of Health and Environmental Research, as an overview of the Human Genome Project.