Chain terminators, deoxynucleotides

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. 

Enzymatic techniques can employ a variety of DNA or RNA polymerases to add controlled amounts of modified nucleotides to an existing stand. However, the most common procedures utilize either DNA polymerase I or terminal deoxynucleotide transferase. The polymerase is used with a template to add modified nucleoside triphosphates to the end of a DNA molecule or to various sites within the middle of a sequence. The terminal transferase can add modified monomers to the 3 end of a chain without a template. 

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. 

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... 

In DNA and RNA, the chain of phosphodiester links and sugar rings is known as the phosphodiester backbone the bases may be regarded in both cases almost as side-chains . By convention, DNA or RNA chains begin at the 5 -end (i.e., where carbon atom C-5 of the terminal residue is not involved in a phosphodiester link) and terminate at the 3 -end (where carbon atom C-3 is not involved in a phosphodiester link). Each chain is therefore said to run by convention from S to S (5 —>-3 ). Several shorthand conventions are used to describe the sequences of deoxynucleotide or nucleotide residues in DNA and RNA respectively. These include the Fischer, linear alphabetic and condensed alphabetic conventions that draw upon the letter codes for bases and deoxynucleosides or nucleosides as described previously (Figure 1.59). 

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