T

Denature and separate daughter strands by electrophoresis

▲ EXPERIMENTAL FIGURE 9-23 Cloned DNAs can be sequenced by the Sanger method, using fluorescent-tagged dideoxyribonucleoside triphosphates (ddNTPs). (a)

A single (template) strand of the DNA to be sequenced (blue letters) is hybridized to a synthetic deoxyribonucleotide primer (black letters). The primer is elongated in a reaction mixture containing the four normal deoxyribonucleoside triphosphates plus a relatively small amount of one of the four dideoxyribonucleoside triphosphates. In this example, ddGTP (yellow) is present. Because of the relatively low concentration of ddGTP incorporation of a ddGTP and thus chain termination, occurs at a given position in the sequence only about 1 percent of the time. Eventually the reaction mixture will contain a mixture of prematurely terminated

(truncated) daughter fragments ending at every occurrence of ddGTP (b) To obtain the complete sequence of a template DNA, four separate reactions are performed, each with a different dideoxyribonucleoside triphosphate (ddNTP). The ddNTP that terminates each truncated fragment can be identified by use of ddNTPs tagged with four different fluorescent dyes (indicated by colored highlights). (c) In an automated sequencing machine, the four reaction mixtures are subjected to gel electrophoresis and the order of appearance of each of the four different fluorescent dyes at the end of the gel is recorded. Shown here is a sample printout from an automated sequencer from which the sequence of the original template DNA can be read directly. N = nucleotide that cannot be assigned. [Part (c) from Griffiths et al., Figure 14-27.]

DNA polymerase, once incorporated they cannot form a phosphodiester bond with the next incoming nucleotide triphosphate. Thus incorporation of a ddNTP terminates chain synthesis, resulting in a truncated daughter strand.

Sequencing using the Sanger dideoxy chain-termination method begins by denaturing a double-stranded DNA fragment to generate template strands for in vitro DNA synthesis. A synthetic oligodeoxynucleotide is used as the primer for four separate polymerization reactions, each with a low concen-

10 Ways To Fight Off Cancer

10 Ways To Fight Off Cancer

Learning About 10 Ways Fight Off Cancer Can Have Amazing Benefits For Your Life The Best Tips On How To Keep This Killer At Bay Discovering that you or a loved one has cancer can be utterly terrifying. All the same, once you comprehend the causes of cancer and learn how to reverse those causes, you or your loved one may have more than a fighting chance of beating out cancer.

Get My Free Ebook


Post a comment