Concepts

DNA can be rapidly sequenced by the dideoxy method, in which ddNTPs are used to terminate DNA synthesis at specific bases. Automated sequencing methods allow tens of thousands of base pairs to be read in just a few hours.

rt A single-stranded DNA fragment whose base sequence is to be determined (the template) is isolated.

^ Each of the four ddNTPs is tagged with a fluorescent dye, and the Sanger sequencing reaction is carried out.

The fragments that end in the same base have the same colored dye attached.

^ The products are denatured, and the DNA fragments produced by the four reactions are mixed and loaded into a single well on an electrophoresis gel. The fragments migrate through the gel according to size,..

. and the fluorescent dye on the DNA is detected by using a laser beam and detector.

^ Each fragment appears as a peak on the computer printout; the color of the peak indicates which base the peak represents.

yn The sequence information is read directly into the computer, which converts it into the complementary—target—sequence.

ddCTP ddGTP ddTTP ddATP

ddCTP ddGTP ddTTP ddATP

dNTPs r dNTPs

Template strand

Primer (sequence known)

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