Box DNA sequencing

Figure 8.6 shows a DNA sequence electropherogram from a capillary electrophoresis DNA sequencer. In the chain-termination method (also called the Sanger method after its inventor Frederick Sanger), a DNA polymerase and oligonucleotide primer are used to copy a DNA template sequence in the presence of a low concentration of dideoxynucleotides. The dideoxynucleotides lack a 3' hydroxyl (-OH) group necessary for continued 5'-to-3' DNA synthesis and therefore the replicating copy ends whenever a dideoxynucleotide is added by the polymerase. The dideoxynucleotides also carry a radioactive or molecular dye marker. When the population of DNA copies is separated by size using electrophoresis, the molecular label is used to identify the type of dideoxynucleotide base (an A, T, C, or G) that terminated the replicating copy. Modern DNA sequencers use laser light to detect one of four molecular dyes attached to each dideoxynucleotide during electrophoresis.

In Fig. 8.6 the y axis represents the intensity of molecular dye signal measured as DNA fragments pass by a laser reader. Each line style represents the intensity of a different color light detected by the laser. Each of the four light colors represents a different dideoxynucleotide. See the text web page for a full-color version of this figure.

0 0

Post a comment