Dideoxynucleotides

DNA polymerases normally use 3 -deoxynucleotide triphosphates as substrates for polymerization. Given an adequate concentration of substrate, DNA polymerase synthesizes a long strand of new DNA complementary to the substrate. The use of this reaction for sequencing DNA depends on the inclusion of a single 2/3 -dideoxynucleoside triphosphate (ddNTP) in each of four polymerization reactions. The dideoxynucleotides ate incorporated normally in the chain in response to a complementary residue in the template. Because no 3 -OH is available for further extension, polymerization is... 

Because dideoxynucleotides lack 3 -OH groups, these nucleotides cannot serve as acceptors for 5 -nucleotide addition in the polymerization reaction, and thus the chain is terminated where they become incorporated. The concentrations of the four deoxynucleotides and the single dideoxynucleotide in each reaction mixture are adjusted so that the dideoxynucleotide is incorporated infrequently. Therefore, base-specific premature chain termination is only a random, occasional event, and a population of new strands of varying length is synthesized. Four reactions are run, one for each dideoxynucleotide, so that termination, although random, can occur everywhere in the sequence. In each mixture, each newly synthesized strand has a dideoxynucleotide at its 3 -end, and its presence at that position demonstrates that a base of that particular kind was specified by the template. A radioactively labeled dNTP is included in each reaction mixture to provide a tracer for the products of the polymerization process. 

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. 

Automated methods for doing this are available that can sequence >500 bp in one run and automatically read out the sequence. Each type of dideoxynucleotide product is marked with a different color so that all four sequencing reactions can be run in one lane of the gel. These automated methods are being used to complete the sequence of the whole human genome. 

A sample of the DNA was reacted with DNA polymerase and each of the nucleotide mixtures (in an appropriate buffer) listed below. Dideoxynucleotides (ddNTPs) were added in relatively small amounts. 

Split the sample into four tubes plus one of the four dideoxynucleotides. 

Synthesis proceeds until the dideoxy-nucleotide is incorporated into a DNA strend. DNA terminating in a dideoxynucleotide cannot be elongated because it lacks a 3 -OH, which is required for chain elongation. 

Detailed structures of several DNA polymerases are available, including those with double- or single-stranded DNA bound.31-35 Site-directed mutagenesis (Chapter 14) has been used to identify residues that are in the active sites for polymerization and exonuclease activity.36,37 Mutants that are defective in the exonuclease activity are used for the production of stable complexes with single-stranded DNA or single-stranded 3 ends of duplexes bound in the exonuclease site.31,33 2, 3 -Dideoxynucleotides (ddNTPs) that lack the nucleophilic 3 -OH have been used to make templates that are inert so that productive ternary enzyme DNA ddNTP complexes may be observed.34,35,38... 

Chemistry of anti-HIV 2,3-dideoxynucleotides and their analogs 92CRV1745. 

Lane 4 When all the deoxynucleotides were provided, but no dideoxynucleotide, a single labeled product formed 5 -primer-TAATGCGTTCCTGTAATCTG. This will appear as a single thick band at the top of the gel. 

Because there is only a small amount of dideoxynucleotide present, it is incorporated only occasionally. However, wherever it is incorporated into the growing piece of DNA, the polymerization is terminated because there is no 3 -hydroxy group to which the next nucleotide can be attached. This results in a series of DNA fragments of different lengths, each terminated at the base that was added as its dideoxy derivative. The synthesis of DNA fragments using dideoxycytosine triphosphate is outlined in Figure 27.5. 

---