Dideoxynucleotide labeling

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. 

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. 

Four separate reactions are set up in the tips of drawn out capillary tubes. Each reaction mixture contains the four deoxynucleotide triphosphates one of which in each case is present in limiting amounts corresponding to the dideoxynucleotide added. In the protocol described a-[32P]-dATP is used as the label but, with the appropriate changes in the compositions of the different dNTP mixes, any of the a-pP] labelled nucleotides could be used. The extension reaction is catalysed using DNA polymerase (Klenow subfragment)... 

Fig. 4.12. Principle of the dideoxynucleotide chain-termination procedure. Primer ( ) is annealed to the single-stranded template at a site adjacent to the cloned sequence. Chain extension in the presence of the competing dideoxynucleotide results in the random incorporation of that nucleotide at the appropriate sites in the extended product. The mixture of labelled chain-terminated products are fractionated according to size by electrophoresis on a denaturing acrylamide gel and the ladder of products revealed by autoradiography.

Each reaction mixture contains the heteroduplex, DNA polymerase, a mixture of the four dNTP species (N = G, A, T, or C), and a small amount of one of the four dideoxynucleotide triphosphates (ddNTP), where no 3 -OH group is present. The ratio of dNTP/ddNTP is high, about 1200 1. DNA polymerase sequentially extends the primer with bases complementary to those present on the opposite strand, but stops when one of the ddNTP species is incorporated. This yields a mixture of fragment lengths, all labeled, where polymerization has stopped at one... 

Add DMA polymerase, nucleotide mixture (dNTP), and labeled terminator dideoxynucleotides (ddA, ddG, ddT and ddC ... 

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... 

Results are read out with the LS4 scanner (GenomicSolutions Ltd.). The scanner provides four lasers (488, 532, 594 and 633 nm) for simultaneous detection of all four labeled dideoxynucleotides. Data processing to obtain easy to handle output formats is performed with the GeneTAC LS4 software. 

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... 

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