Dideoxynucleotides
Encyclopedia
Dideoxynucleotides, or ddNTPs, are nucleotide
s lacking a 3'-hydroxyl (-OH) group on their deoxyribose
sugar. Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks hydroxyl groups at both its 2' and 3' carbons. The lack of this hydroxyl group means that, after being added by a DNA polymerase
to a growing nucleotide chain, no further nucleotides can be added as no phosphodiester bond
can be created based on the fact that deoxyribonucleoside triphosphates (which are the building blocks of DNA) allow DNA chain synthesis to occur through a condensation reaction between the 5' phosphate (following the cleavage of pyrophospate) of the current nucleotide with the 3' hydroxyl group of the previous nucleotide. The dideoxyribonucleotides do not have a 3' hydroxyl group, hence 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 of DNA sequencing
, which was developed by Frederick Sanger
in 1977.
Dideoxynucleotides are useful in the sequencing of DNA in combination with electrophoresis
. A DNA sample that undergoes PCR (polymerase chain reaction
) in a mixture containing all four deoxynucleotides and one dideoxynucleotide will produce strands of length equal to the position of each base of the type that complements the type having a dideoxynucleotide present. That is, each nucleotide base of that particular type has a probability of being bonded to not a deoxynucleotide but rather a dideoxynucleotide, which ends chain elongation. Thus, if the sample then undergoes electrophoresis, there will be a band present for each length at which the complement
of the dideoxynucleotide is present. It is now common to use fluorescent dideoxynucleotides such that each one of the four has a different fluorescence that can be detected by a sequencer; thus only one reaction is needed.
Nucleotide
Nucleotides are molecules that, when joined together, make up the structural units of RNA and DNA. In addition, nucleotides participate in cellular signaling , and are incorporated into important cofactors of enzymatic reactions...
s lacking a 3'-hydroxyl (-OH) group on their deoxyribose
Deoxyribose
Deoxyribose, more, precisely 2-deoxyribose, is a monosaccharide with idealized formula H---3-H. Its name indicates that it is a deoxy sugar, meaning that it is derived from the sugar ribose by loss of an oxygen atom...
sugar. Since deoxyribose already lacks a 2'-OH, dideoxyribose lacks hydroxyl groups at both its 2' and 3' carbons. The lack of this hydroxyl group means that, after being added by a DNA polymerase
DNA polymerase
A DNA polymerase is an enzyme that helps catalyze in the polymerization of deoxyribonucleotides into a DNA strand. DNA polymerases are best known for their feedback role in DNA replication, in which the polymerase "reads" an intact DNA strand as a template and uses it to synthesize the new strand....
to a growing nucleotide chain, no further nucleotides can be added as no phosphodiester bond
Phosphodiester bond
A phosphodiester bond is a group of strong covalent bonds between a phosphate group and two 5-carbon ring carbohydrates over two ester bonds. Phosphodiester bonds are central to all known life, as they make up the backbone of each helical strand of DNA...
can be created based on the fact that deoxyribonucleoside triphosphates (which are the building blocks of DNA) allow DNA chain synthesis to occur through a condensation reaction between the 5' phosphate (following the cleavage of pyrophospate) of the current nucleotide with the 3' hydroxyl group of the previous nucleotide. The dideoxyribonucleotides do not have a 3' hydroxyl group, hence 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 of DNA sequencing
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
, which was developed by Frederick Sanger
Frederick Sanger
Frederick Sanger, OM, CH, CBE, FRS is an English biochemist and a two-time Nobel laureate in chemistry, the only person to have been so. In 1958 he was awarded a Nobel prize in chemistry "for his work on the structure of proteins, especially that of insulin"...
in 1977.
Dideoxynucleotides are useful in the sequencing of DNA in combination with electrophoresis
Electrophoresis
Electrophoresis, also called cataphoresis, is the motion of dispersed particles relative to a fluid under the influence of a spatially uniform electric field. This electrokinetic phenomenon was observed for the first time in 1807 by Reuss , who noticed that the application of a constant electric...
. A DNA sample that undergoes PCR (polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
) in a mixture containing all four deoxynucleotides and one dideoxynucleotide will produce strands of length equal to the position of each base of the type that complements the type having a dideoxynucleotide present. That is, each nucleotide base of that particular type has a probability of being bonded to not a deoxynucleotide but rather a dideoxynucleotide, which ends chain elongation. Thus, if the sample then undergoes electrophoresis, there will be a band present for each length at which the complement
Complementary DNA
In genetics, complementary DNA is DNA synthesized from a messenger RNA template in a reaction catalyzed by the enzyme reverse transcriptase and the enzyme DNA polymerase. cDNA is often used to clone eukaryotic genes in prokaryotes...
of the dideoxynucleotide is present. It is now common to use fluorescent dideoxynucleotides such that each one of the four has a different fluorescence that can be detected by a sequencer; thus only one reaction is needed.