Nucleotide sequencing dideoxynucleotide chain termination method

Cloning and screening of the genomic DNA were performed as described elsewhere (7). The nucleotide sequence of the genomic clone was determined by the dideoxynucleotide chain-termination method (8). 

FIGURE 12.3 The chain termination or dideoxy method of DNA sequencing, (a) DNA polymerase reaction, (b) Structure of dideoxynucleotide. (c) Four reaction mixtures with nucleoside triphosphates plus one dideoxynucleoside triphosphate, (d) Electro-phoretogram. Note that the nucleotide sequence as read from the bottom to the top of the gel is the order of nucleotide addition carried out by DNA polymerase. 

The newly synthesized DNA will be complementary to the sequence of the foreign or inserted DNA. DNA synthesis, catalyzed by DNA polymerase, requires the presence of all of the deoxynucleotide bases. The Sanger method utilizes modified bases, called dideoxynucleotides, which lack the 3 -hydroxy on the sugar residue that normal nucleotides have. When DNA polymerase incorporates a dideoxynu-cleotide into a growing strand of DNA, the strand terminates immediately thereafter. Chain termination occurs because the dideoxy nucleotide lacks the 3 -hydroxy... 

Fig. 17.7. The Sanger method. (A). A reaction mixtures contain one of the dideoxynucleotides, such as ddATP, and some of the normal nucleotide, dATP, which compete for incorporation into the growing polypeptide chain. When a T is encountered on the template strand (position 10), some of the molecules will incorporate a ddATP, and the chain will be terminated. Those that incorporate a normal dATP will continue growing until position 15 is reached, where they will incorporate either a ddATP or the normal dATP. Only those that incorporate a dATP will continue growing to position 17. Thus, strands of different length from the 5 end are produced, corresponding to the position of a T in the template strand. (B). DNA sequencing by the dideoxynu-cleotide method. Four tubes are used. Each one contains DNA polymerase, a DNA template hybridized to a primer, plus dATP, dGTP, dCTP, and dTTP. Either the primer or the nucleotides must have a radioactive label, so bands can be visualized on the gel by autoradiography. Only one of the four dideoxyribonucleotides (ddNTPs) is added to each tube. Termination of synthesis occurs where the ddNTP is incorporated into the growing chain. The template is complementary to the sequence of the newly synthesized strand.

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