2 ,3 -dideoxynucleoside triphosphate

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... 

Nucleotides 2 -deoxynucleoside triphosphates (dNTPs) are purchased from Sigma (St. Louis, MO), and the 2, 3 -dideoxynucleoside triphosphates (ddNTPs) from Boehringer-Mannheim Biochemicals (Mannheim, Germany)... 

In the dideoxy method a small piece of DNA called a primer, labeled at the 5 -end with is added to the restriction fragment whose sequence is to be determined. Next, the four 2 -deoxyribonucleoside triphosphates are added as well as DNA polymerase, the enzyme that adds nucleotides to a strand of DNA. In addition, a small amount of the 2, 3 -dideoxynucleoside triphosphate of one of the bases is added to the reaction mixture. A 2, 3 -dideoxynucleoside triphosphate has no OH groups at the 2 -and 3 -positions. 

The key to the chain termination method is the addition of a 2, 3 -dideoxynucleoside triphosphate (ddNTP) to the synthesizing medium ... 

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. 

The Sanger dideoxynucleoside method of sequencing DNA. (a) A suitable template is chosen, and the primer is chosen so that DNA synthesis begins at the point of interest. The primer is radioactively labeled. In addition to the template-primer complex the reaction mixture contains all four radioactive deoxyribonucleoside triphosphates and small amounts of a single dideoxynucleoside triphosphate. The dideoxy compound serves as a chain terminator. 

Four incubation mixtures are set up, each containing the DNA template, a specific DNA primer, E. coli DNA polymerase I and all four deoxyribo-nucleoside triphosphates (dNTPs). In addition, each mixture contains a different dideoxynucleoside triphosphate analog, ddATP, ddCTP ddGTP or ddTTP. Incorporation of a dideoxy analog prevents further elongation and so produces a chain termination extension product. The products are electrophoresed on a polyacrylamide gel and the DNA sequence read from the band pattern produced. 

To determine the sequence of the template DNA, four versions of the polymerization are run, each with a different dideoxynucleoside triphosphate. The reactions are analyzed using gel electrophoresis. In this technique the mixtures of synthesized fragments from each reaction are placed in separate lanes at the top of a layer of a polyacrylamide gel and a voltage is applied between the top and the bottom. Smaller molecules move faster than larger molecules, so they appear closer to the bottom of the gel... 

Taken all in all the plus and minus method is a powerful technique. However, it does have its limitations and the more recent developments using the dideoxynucleoside triphosphates as specific chain inhibitors have much improved both the speed and accuracy of the method and will usually be the method of choice for primed synthesis sequencing. This is described in the next chapter. 

Direct enzymatic method for sequencing double-stranded restriction fragments using dideoxynucleoside triphosphates (Maat and Smith, 1978)... 

Fig. 3.12. Autoradiograph of sequencing gels prepared using, he sim dideoxy-DNA polymerase method for sequencing 5 -end labelled DNA r T The sequences shown were derived from two 5 -end labelled T r agments first 22 nucleotides were run off the gel. Channel B shows the olT 1 The incubation with Klenow polymerase alone and Channel 4 is the natte 8 "erated bV the presence of all four dideoxynucleoside triphosphates. (From Hindfoy an T...

Chom extension with Klenow polymerase in the presence of one of the dideoxynucleoside triphosphates (4 reactions) and a -[ pJdATP... 

DNA Polymerase I, Klenow sub-fragment from Boehringer Mannheim, in a glycerol solution at a concentration of about 1 unit Dideoxynucleoside triphosphates are obtained either from P-L-Biochemicals Inc. or from Collaborative Research. The octanucleotide EcoRI linker 5 -GGAATTCC was from Col-... 

Stocks of 10 mM dideoxynucleoside triphosphates are kept frozen in water (or 5 mM Tris-Cl pH 8.0, 0.1 mM EDTA). Working stocks are prepared by appropriate dilution (with water). As a first approximation prepare ... 

Add 1 pi of the following four dideoxynucleoside triphosphate solutions to each of the 6-jjl1 aliquots ... 

Sanger sequencing is the most common technique used in molecular laboratories to detect the exact nucleotide composition of PCR-amplified DNA fragments. It uses chain-terminating dideoxynucleoside triphosphates... 

The dideoxynucleosides do not have a hydroxyl group on either the 2 - or 3 -car-bon. They can be converted to dideoxynucleoside triphosphates in cells and, like ZDV, terminate chain growth when incorporated into DNA. In the case of the dideoxynucleosides, chain termination results from the absence of a hydroxyl group on the 3 carbon. The HIV virus mutates very rapidly (mostly because reverse transcriptase lacks 3 to 5 exonuclease activity, the proofreading activity) and frequently develops resistance to one or more of these drugs. Therefore, it is recommended that AIDS patients take a number of drugs, including more than one reverse transcriptase inhibitor. 

The 2, 3 -dideoxynucleosides can be used as reagents to inhibit DNA replication. These analogs must be converted to dideoxynucleoside triphosphates in order to have a measurable effect on DNA synthesis. When incorporated into a growing DNA chain, a single dideoxyribonucleoside residue can effectively block subsequent chain extension. 

In order to interact with their target enzyme, the reverse transcriptase, pharmacologically effective levels of 2, 3 -dideoxynucleoside-5 -triphosphates have to be generated. This implies that the candidate 2, 3 -dideoxynucleoside has to enter the cell, using the nucleoside transport mechanisms or by passive diffusion. The 2, 3 -dideoxynucleoside must also be transformed to the corresponding 5 -triphosphate by cellular kinases, because HIV does not appear to encode for nucleoside kinases. The efficiency of this phosphorylation strongly depends on the type of compound and the nature of the cells. The susceptibility of the nucleosides and nucleotides involved in this process to metabolic enzymes such as deaminases, hydrolases and phosphatases is another important factor to reach and maintain effective levels of dideoxynucleoside triphosphates. 

In molecular biology, TDT is used to effect the addition of complementary homopolymeric tails to vector and complementary DNA (cDNA) obtained from a mature mRNA template by reverse transcription and PCR amplification. An alternative application of TDT involves labehng of the 3 termini of DNA fragments with a P-labeled dNTP [36], a dideoxynucleoside triphosphate (ddNTP) [37], or a ribonucleoside triphosphate (rNTP) [38]. 

Many different fluorophores have been incorporated into oligonucleotides, covering applications such as sequencing, fluorescence detection and FRET dyes. Several sets of dideoxynucleoside triphosphates have been reported for... 

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