Dideoxynucleotide triphosphate

One p of a different dideoxynucleotide triphosphate solution is then added to each capillary. The concentrations of the stock solutions of each ddNTP which give similar extensions are ... 

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 1.0 p,l of Tag DNA polymerase (5 U/ ji,l) to the mix. Add 4 xl of the resulting mix to each tube containing 2 p,l of d/ddNTP (deoxy-dideoxynucleotide triphosphate) Mix. Place the reaction tubes in a thermal cycler that has been preheated to 95° and start the cycling program as follows ... 

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... 

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. 

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. 

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)... 

Terminal deoxynucleotidyl transferase (TdT) polymerizes DNA by the same mechanism as DNA polymerases but lacks a requirement for a template DNA strand to direct synthesis (27, 28). TdT prefers single-stranded DNA or duplex DNA containing 3 protruding ends, and is often used in the synthesis of a homopolymeric tail. Nucleotide incorporation is influenced by the divalent cation in the reaction Purine nucleotides are incorporated preferentially in the presence of Mg +, while incorporation of pyrimidine nucleotides is favored in the presence of Co. The use of nucleotide analogs (dideoxynucleotides or cor-dycepin triphosphate) results in the incorporation of a single nucleotide (29). 

Each of the four tubes containing the DNA, enzymes, and an excess of the nucleotides required for replication will also have a small amount of one of the four dideoxynucleotides. In the tube that receives dideoxyadenosine triphosphate (ddA), for example, DNA synthesis will begin. As replication proceeds, either the standard nucleotide or ddA will be incorporated into the growing strand. Since the standard nucleotide is present in excess, the dideoxynucleotide will be incorporated infrequently and randomly. This produces a family of DNA fragments that terminate at the location of one of the deoxyadenosines in the molecule. 

For many years, the standard approach was to set up four simultaneous reactions with equivalent, defined concentrations of complementary DNA strand, primer and four de-oxynucleotide triphosphate (dNTP) substrates. Each reaction differed only in having a low mol per cent of a single radioactively labelled dideoxynucleotide (ddNTP) substrate (either ddATP, ddGTP, ddCTP or ddTTP). These ddNTPs possess neither l nor 3 hydroxyl group and therefore prevented further DNA polymerisation whenever they were incorporated into... 

The inhibitors of DNA polymerases have been found among dNTP derivates modified in the base (2-substituted dATP and I P-substituted dGTP analogs, derivatives with altered base-pairing specificity), derivates modified in the sugar (arabinonu-cleotides, 2, 3 -dideoxynucleotides, acyclonucleotides) and derivates modified in the triphosphate group (for example, phosphorothioates of dATP) [31-33]. 

RNA sequencing can he directly performed from dsDNA templates cloned downstream of a phage promoter, T7, T3, or SP6 (30,82). Transcription reactions are performed in the presence of a chain terminator, 3 -deoxynucIeoside 5 -triphosphate, in a way similar to the dideoxynucleotide sequencing of DNA. The difference is that the DNA sequencing by RNA transcription obviates the need to prepare ssDNA templates or to use sequencing primers. 

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