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Dideoxyribonucleoside triphosphates (ddNTPs) are used during DNA sequencing because they are the building blocks of DNA. During DNA sequencing, the DNA is cut into smaller fragments, and then the ddNTPs are added to the fragments. The ddNTPs attach to the fragments, and then the fragments are run through a sequencing machine. The sequencing machine reads the sequence of the ddNTPs attached to the fragments, which allows the DNA sequence to be determined.

Dideoxynucleotide

DNA polymerase strand elongation inhibitor

Molecular structure of 2′,3′-dideoxyadenosine triphosphate (ddATP)

Dideoxynucleotides are chain elongation inhibitors. DNA polymeraseused in Sanger method for DNA sequencing. They are also known as 2′, 3′ because both the 2′ and 3′ positions on the ribose lack hydroxyl groupsand are abbreviated as ddNTPs (ddGTP, ddATP, ddTTP and ddCTP).

Role in the Sanger method

Inhibition of a nucleophilic attack due to the absence of the 3′-OH group

The Sanger method is used to amplify a target segment of DNA so that the DNA sequence can be accurately determined. The incorporation of ddNTPs into the reaction valves is simply used to terminate the synthesis of a growing DNA strand, resulting in partially replicated DNA fragments. This is because DNA polymerase requires the 3′ OH group of the growing chain and the 5′ phosphate group of the incoming dNTP to create a phosphodiester bond. Sometimes the DNA polymerase will incorporate a ddNTP and the absence of the 3′ OH group will stop the process. condensation reaction between the 5′ phosphate (following the cleavage of pyrophosphate) of the incoming nucleotide with the 3′ hydroxyl group of the previous nucleotide on the growing strand. This condensation reaction would normally occur with the incorporation of a dNTP not modified by DNA polymerase. In simpler terms, nucleophilic attack on the 3’OH group leads to the addition of a nucleotide in a growing chain. The absence of the 3′ hydroxyl group inhibits the occurrence of this nucleophilic attack, making it impossible for the DNA polymerase to continue with its function.

This discovery led to their apt name “chain terminating nucleotides”. Dideoxyribonucleotides lack a 3′ hydroxyl group, so no further chain elongation can occur once this dideoxynucleotide is on the chain. This can lead to the termination of the DNA sequence. Thus, these molecules form the basis of the dideoxy chain termination method in DNA sequencingthat was reported by Frederick Sanger and his team in 1977 as an extension of their earlier work. Sanger’s approach was described in 2001 as one of two fundamental methods for sequencing DNA fragments (the other being the Maxam–Gilbert Method), but Sanger’s method is “the most widely used and the method used by most automated DNA sequencers”. Sanger won his second Nobel Prize in Chemistry in 1980, sharing it with walter gilberto (“for his contributions to the determination of base sequences in nucleic acids”) and with Paulo Berg (“for his fundamental studies of the biochemistry of nucleic acids, with particular attention recombinant DNA“), and discussed the use of dideoxynucleotides in his Nobel lecture.

DNA sequencing

Dideoxynucleotides are useful in DNA sequencing in combination with electrophoresis. A DNA sample that undergoes PCR (polymerase chain reaction) in a mixture containing all four deoxynucleotides and a dideoxynucleotide will produce chains of length equal to the position of each base of the type that complements the type that has a dideoxynucleotide present. The taq polymerase used in PCR favors ddGNTP, which has been a pattern observed in several studies. That is, each nucleotide base of that particular type has a chance of being attached not to a deoxynucleotide but to a dideoxynucleotide, which ends chain elongation. Therefore, if the sample is subjected to electrophoresis, there will be a band present for each length at which the complement of dideoxynucleotide is present. Today it is common to use fluorescent dideoxynucleotides so that each of the four has a different fluorescence that can be detected by a sequencer; thus, only one reaction is required.

Production

In a patented method, Tetrahydrofuran was used in a solvent containing 50 g (0.205 mol) of uridine (or, presumably, any unprotected nucleoside), 112 ml (1.03 mol) of methyl orthoformate and 10 g (52.6 mmol) of paratoluenesulfonic acid at room temperature with stirring. After stirring at room temperature for 24 hours, the reaction mixture was poured into an aqueous sodium bicarbonate solution followed by extraction with chloroform 5 times. The extract was dried over sodium sulfate and concentrated to give 49.5 g (0.173 mol) of 2′,3′-o-methoxymethylideneuridine (yield, 84.5%). After this reaction, the example product of uridine 2′,3′-o-methoxymethylideneuridine is then dissolved in 50 ml of acetic anhydride at room temperature with stirring. The solution was heated to 140°C and kept at this temperature for 5 hours under solvent reflux. After cooling to room temperature, the solvent was distilled off under reduced pressure, 50 ml of water were added, and the mixture was extracted 3 times with 100 ml of chloroform. The extract was concentrated under reduced pressure, 30% ammonia and water to obtain an 82.3% yield of 2′,3′-dideoxy-2′,3′-didehydrouridine. This example product is then hydrated to remove the double bond by dissolving it in methanol (10 ml) containing a catalyst (5% wet palladium on carbon) (400 mg) in an atmosphere of hydrogen for 1 h to obtain the resulting dideoxynucleoside (2′,3′-dideoxyuridine in this case). The dye coretide to be used will likely occur by treatment with a phosphorylating enzyme and biotinylation and reaction of the biotinylated substance with the dye. It is possible that an immediate reaction with the dye will also occur, but the extension of the arm increases the efficiency in the case of using a mutated form of DNA polymerase

References

External Links


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Search keywords Why Are Dideoxyribonucleoside Triphosphates Used During Dna Sequencing

1. DNA Sequencing
2. Sanger Sequencing
3. DNA Polymerase
4. Chain Termination
5. Dideoxynucleotide Triphosphates
6. DNA Replication
7. Fluorescent Labels
8. Primer Extension
9. Maxam-Gilbert Method
10. DNA Ligation
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