Sanger dideoxynucleotide sequencing

Fig. 6.22. The Sanger DNA-sequencing procedure, (a) The Sanger dideoxynucleotide sequencing reaction with 32p. abeled primers, (b) Acrylamide gel separation of the labeled fragments. (Adapted from Watson JD, Gilman M, Witkowski J, et al. Recombinant DNA, 2nd Ed. New York Scientific American Books, W.H. Freeman and Company, 1992 with permission.)...

Figure 26-16 Sanger dideoxynucleotide sequencing of a sample oligonucleotide, called the template strand. The attached known primer sequence tells the enzyme DNA polymerase where to start replication. Replication stops when a 2, 3 -dideoxyribonucleotide n the experiment shown, it is ddA) is added to the growing chain. Eiectrophoresis of the resulting mixture reveals the length of the fragments with the corresponding end nucleotide and, therefore, the position of this nucleotide in the replicate strand and its complement in the template strand.

Be familiar with DNA sequencing using the Sanger dideoxynucleotide method. 

Base-modified pyrophosphorylated nucleosides. A major contribution to advances in DNA sequencing has been achieved by a team of researchers from Columbia University. They developed a method which combined the Sanger dideoxynucleotide terminating reaction process to that of DNA sequencing by synthesis on a solid surface during polymerase chain reaction. The latter approach allows for deciphering many DNA... 

One sequencing technique is the Sanger method (developed by Fred Sanger) which uses dideoxynucleotides that stop chain elongation at the site of their incorporation. The four dideoxynucleotide substrates are labeled with... 

Recendy, an automated version of the Sanger method has become available. Instead of using radiolabeled primers, it uses fluorescent tagged dideoxynucleotides. Because each dideoxy analogue fluoresces a different color, the entire procedure is carried out in a single test tube. Afterwards the reaction products are loaded and run on a single electrophoresis gel. After a detector scans the gel, a computer determines the sequence of the colored bands (Figure 17G). 

Primer and template in Sanger sequencing. The cartoon, panel 1, shows a long single strand of DNA (labeled "template") annealed to a primer that is complementary in sequence to part of the template. DNA polymerase has already synthesized a "new strand" of DNA, starting from the 30 end of the primer and continuing along the template strand until a dideoxynucleotide was incorporated. The polymerase is now stopped as there is no 30-OH... 

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.

EXAMPLE 3.22 How does a dideoxynucleotide terminate the chain extension reaction in the Sanger method of DNA sequencing ... 

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... 

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