Dideoxy nucleotides

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. 

The primer extension assay is similar to the dideoxy method commonly used in sequence analysis. In the primer extension assay, a region containing the mutation to be assayed is amplified in a first PCR reaction. A specific primer, which is designed to anneal directly upstream of the base which is the site of a known mutation, is then used in a second reaction. Radioac-tively labeled dideoxy nucleotides corresponding to either the normal or the mutant base at the potential mutation site are added in separate tubes. During the reaction, the labeled nucleotide added to the 3 end of the primer will depend on the sequence at the potential mutation site. If the normal sequence is present at the potential mutation site, only the reaction containing the normal labeled dideoxy nucleotide will produce a labeled primer. Conversely, if the mutant sequence is present at the mutation site, only... 

Stocks of 4 mM or 10 mM dideoxy-nucleotide triphosphates (P.L. Biochemicals or Collaborative Research) are kept frozen in water. 

In one experiment (Hindley, J., unpublished data) a set of dideoxy-nucleotide chain extension reactions, using an identical primed-template, was carried out at 14, 20 and 29°C respectively and the reaction products analysed on a standard thin sequencing... 

The dideoxy method of Sanger for sequencing DNA involves the copying of a single strand of the DNA by a DNA polymerase (DNA polymerase I was originally used) to yield new strands (radioactively labeled for identification purposes), which are terminated at certain positions through the incorporation of a dideoxy nucleotide at the 3 end. A short primer, complementary to a sequence at one end of the single-strand template. 

Electrophoresis is followed by laser activation of the fluorescent dideoxy nucleotides, and detection to distinguish the colours... 

Figure 5-21. DNA sequencing by the Sanger-dideoxy method. Single or double stranded DNA is used as a template for a primer-dependent DNA polymerase extension, which is carried out in four separate reaction mixes. In addition to DNA polymerase and the four dNTPs (including an a-radiolabelled nucleoside triphosphate), each reaction mix contains a small proportion of one dideoxy nucleotide derivative, ddATP,...

Figure 5.5 Fluorescence detection of oligonucleotide fragments produced by the dideoxy method. A sequencing reaction is performed with four chain-terminating dideoxy nucleotides, each labeled with a tag that fluoresces at a different wavelength (e.g., red for T). Each of the four colors represents a different base in a chromatographic trace produced by fluorescence measurements at four wavelengths. [After A. J. F. Griffiths et al., An Introduction to Genetic Analysis, 8th ed. (W. H. Freeman and Company, 2005).]...

Dideoxy nucleotides are used to terminate DNA synthesis. Multiple reactions are run with a different dideoxy nucleotide in each reaction mix. The reactions produce a series of DNA fragments of different length that can be run on a gel and the sequence determined by tracking the different length fragments in the lanes with the four different dideoxy nucleotides. 

How do dideoxy nucleotides allow us to sequence DNA The Sanger-Goulson method allows for the determination of a DNA sequence by using dideoxy nucleotides. These nucleotides cause chain termination during DNA synthesis. By running parallel reactions with a dideoxy version of one each of the four deoxynucleotides, we can see a banding pattern on a gel that allows us to read the DNA sequence. 

In more modem methods, each of the dideoxy nucleotides carries a different fluorescent tag molecule, or fluorophor. Thus, all four chain-termination reactions can be carried out in a single sample tube. The different DNA strands are then separated by capillary electrophoresis and detected on the basis of their particular fluorescence. 

As shown in Fig. 8-20 for ddATP, because dideoxy nucleotides have a hydrogen atom and not an OH at the 3 position of the pentose ring, when they are incorporated into a DNA chain that is being synthesized, addition of further bases is not possible. This will occur in only a minority of the DNA chains being synthesized in the solution, because of the trace concentrations of the dideoxy nucleotides. The outcome is a series of fragments of DNA that can be resolved by gel electrophoresis and visualized by autoradiography, exploiting the fact that the primers are radioactive. 

The fundamental strategy was the same, but the process was automated. Each of the dideoxy nucleotides was tagged with a different fluorescent dye. The four separately reacted mixtures were combined and electrophoresced in a single lane using a robotically controlled sequencer, which can analyze 96 samples simultaneously. As each fragment reached the bottom of the electrophoresis lane, its dideoxy nucleotide was identified from its characteristic fluorescence. Automatic sequencers can sequence 5 x 10 bases per day compared to 3 x lO per year using the manual methods described in Fig. 8-21. 

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... 

---