(78)
9. Forklare principet i Sanger (dideoxy) sekentering samt beskrive gelelektrophorese
Devlin, s.285-7, fig. 7.5
Stryer, s.146-7, fig. 6.5
Color atlas of biochemistry, s.252

This method is used for DNA sequencing.

It is based on the random termination of a DNA chain during enzymatic synthesis when the solution is presented to a small amount of the dideoxynucleotide analogs of the four normal nucleotides.

The dideoxynucleotide analogs can be incorporated by DNA polymerase into the growing DNA chain, but they also terminate the chain, since they only have a 3’-H atom instead of the 3’-OH group on the ribose, which is supposed to participate in making a phosphodiester bond with the adjacent nucleotide. Since these analogs are added in small amounts during replication of the template strand, DNA polymerase sometimes incorporates the normal complementary nucleotide, sometimes the complementary analog which randomly terminates the chain.  

The procedure can be performed with autoradiography or fluorescence detection.
 

Autoradiography – the procedure is performed on four reaction mixtures in the same time. In each mixture there is a template DNA, labelled primers, deoxyribonucleoside triphosphates which are radioactively labelled and a small amount of a specific dideoxynucleotide analogs, a different nucleotide in each reaction mixture. The concentration of these analogs is low enough that chain termination takes place only occasionally at all possible complementary sites to the DNA template. If we have a mixture to which we only have added the dideoxy analog of ATP, then it will be inserted randomly terminating the chain where a T was located in the template strand.

That way, we get a lot of fragments with different lengths that correspond with the position of T in the complementary strand. They are all labelled at the 5’-end because of the labelled primer.

 The other reaction mixtures get also fragments with different lengths corresponding to the other bases.

Therefore, four sets of chain-terminated fragments, one for each dideoxy analog undergo electrophoresis which separates them on length bases and the base sequence of the template DNA is read from the audioradiogram of the four lanes.
 

Fluorescence detection – a fluorescent tag is attached to an oligonucleotide priming fragment – a differently coloured one in each of the four reaction mixtures. The reaction mixtures are combined and subjected to electrophoresis together. The separated bands of DNA are then detected by their fluorescence as they emerge from the gel; the sequence of their colours directly gives the base sequence.     

Electrophoresis - procedure for the separation of electrically charged molecule based on their migration in an electric field. The velocity of migration of a molecule is determined by its net charge, its size and shape and the magnitude of the voltage applied. The net charge is determined on the number of negatively and positively charaged residues and the pH of the electrophoresis buffer.

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