AAT Bioquest

Dideoxynucleotides (ddNTPs)

ddNTP are molecules similar in chemical structure to deoxynucleotides (dNTPs), though they lack a 3' hydroxyl group (3'-OH) on their deoxyribose sugar. There are four ddNTPs, so named after their respective nitrogenous bases which are either adenine (ddATP), guanine (ddGTP), cytosine (ddCTP), or thymine (ddTTP).

In a normal dNTP, the 3' hydroxyl group is essential for polymerase-mediated strand elongation in a polymerase chain reaction (PCR). However, without the hydroxyl group no phosphodiester bond can form between the 3' end of the last nucleotide and the 5' end of the next. This means that once a ddNTP is added by a DNA polymerase to a growing nucleotide chain, no further nucleotides can be added to the template and DNA synthesis is stopped. This ability of ddNTPs to effectively inhibit chain elongation is a unique characteristic that has been widely exploited to determine precise DNA sequences in research.

Sanger Sequencing

Sanger sequencing by capillary electrophoresis
An illustration of Sanger sequencing by capillary electrophoresis (figure made in BioRender).
The dideoxy chain-termination method of DNA sequencing, or simply chain-termination sequencing, was developed by Frederick Sanger in 1977. The Sanger sequencing method has been used throughout research to determine the precise makeup of unknown DNA.

During Sanger Sequencing:
  1. The unknown DNA sequence is combined with dNTPs, DNA polymerase and a (sometimes radioactive-linked) primer.
  2. Four reaction mixes are prepared that each contain one of the four separate, fluorescently- or radio-labeled, ddNTPs, and added in small quantities to the reaction mix.
  3. The mix is heated to denature the template DNA, then cooled to allow for primer binding.
  4. The temperature of the reaction is raised again which allows for DNA polymerase to synthesize new DNA. DNA polymerase will continue to add nucleotides from the dNTPs complementary to the template until it adds a ddNTP; at this point, no further nucleotides can be added.

This process is repeated, and once cycling is finished it is nearly guaranteed that a ddNTP will have been incorporated at every single position of the target DNA. Upon analysis, the reaction will contain DNA fragments of various lengths, ending at each of the nucleotide positions in the original template DNA. The tagged ddNTPs can then be used to determine the final nucleotide sequence. Today, principles of Sanger sequencing technology have been automated which has allowed for the increasingly high-throughput ability of processing and results.


Single Base Extension (SBE) Technology

A single nucleotide polymorphism (SNP) is a genomic variant at a single base position in the DNA. SNPs are the most common form of polymorphisms present in the human genome, and can influence health, disease, and even drug response. SNPs occur throughout the genome, in noncoding regions, exons, and introns. The SBE assay is a specific type of genotyping technique that targets certain SNPs within a DNA sequence through the use of ddNTPs.

In essence, the SBE assay can accurately identify adenine/thymine (A/T) and cytosine/guanine (C/G) base incorporation at specific SNP loci. Similar to Sanger sequencing, the SBE assay relies on the extension of a primer, designed to bind one nucleotide upstream of the polymorphic spot via a complementary ddNTP. Subsequent detection of the incorporated ddNTP is performed by mass spectrometry which can reveal the nucleotide base at that exact position on the DNA template strand.

In just a single reaction, this technique can effectively and sensitively type over 30 known loci scattered throughout an organism's genome. SBE technologies have also allowed for the typing of tetra-allelic SNPs, and methods have been adapted to a broad range of applications, including for use in single-cell analysis, diagnosis of monogenic diseases, forensic mitochondrial DNA analysis on human remains, and high-throughput SNP screening.




Table 1. ReadiUse™ ddNTP Terminator Mix

Product Name
Unit Size
17205ReadiUse™ ddNTP Terminator Mix *10 mM*100 nmoles
17206ReadiUse™ ddNTP Terminator Mix *10 mM*10 umoles

Table 2. Available MagaDye™ fluorescent ddNTPs for Sanger sequencing.

Product Name
Ex (nm)
Em (nm)
Unit Size
Cat No.
MagaDye™ 535-ddGTPGuanine503 nm536 nm503 nm5 nmoles17063
MagaDye™ 535-ddGTPGuanine503 nm536 nm503 nm50 nmoles17067
MagaDye™ 561-ddATPAdenine498 nm561 nm498 nm5 nmoles17062
MagaDye™ 561-ddATPAdenine498 nm561 nm498 nm50 nmoles17066
MagaDye™ 588-ddTTPThymine498 nm588 nm498 nm5 nmoles17061
MagaDye™ 588-ddTTPThymine498 nm588 nm498 nm50 nmoles17065
MagaDye™ 613-ddCTPCytosine498 nm614 nm498 nm5 nmoles17060
MagaDye™ 613-ddCTPCytosine498 nm614 nm498 nm50 nmoles17064

Table 3. Building Blocks for Developing Sanger Sequencing Reagents

Product Name
Unit Size
Cat No.
Aminopropargyl ddCTP [5-Propargylamino-2',3'-dideoxycytidine-5'-triphosphate] 10 µmoles17070
Aminopropargyl ddTTP [5-Propargylamino-2',3'-dideoxyuridine-5'-triphosphate] 10 µmoles17072
Aminopropargyl ddATP [7-Deaza-7-Propargylamino-2',3'-dideoxyadenosine-5'-triphosphate]10 µmoles17074
Aminopropargyl ddGTP [7-Deaza-7-Propargylamino-2',3'-dideoxyguanosine-5'-triphosphate]10 µmoles17076
2-Aminoethoxypropargyl ddCTP1 µmoles17080
2-Aminoethoxypropargyl ddTTP1 µmoles17082
2-Aminoethoxypropargyl ddATP1 µmoles17084
2-Aminoethoxypropargyl ddGTP1 µmoles17086
7-Deaza-7-Propargylamino-3'-azidomethyl-dATP50 nmoles17090
5-Propargylamino-3'-azidomethyl-dCTP50 nmoles17091
7-Deaza-7-Propargylamino-3'-azidomethyl-dGTP50 nmoles17092
5-Propargylamino-3'-azidomethyl-dUTP50 nmoles17093
ddCTP [2',3'-Dideoxycytidine-5'-triphosphate] *10 mM in ddH2O*1 µmole17207
ddTTP [2',3'-Dideoxythymidine-5'-triphosphate] *10 mM in ddH2O*1 µmole17208
ddATP [2',3'-Dideoxyadenosine-5'-triphosphate] *10 mM in ddH2O*1 µmole17209
ddGTP [2',3'-Dideoxyguanosine-5'-triphosphate] *10 mM in ddH2O* 1 µmole17210
dATP *100 mM PCR Grade*500 µL17250
dCTP *100 mM PCR Grade*500 µL17252
dTTP *100 mM PCR Grade*500 µL17256

Table 4. Deoxynucleotides (dNTPs) for use in PCR, real-time PCR, and reverse transcription PCR

Unit Size
Cat No.
ReadiUse™ dNTP Mix *10 mM*Water5 mL17200
ReadiUse™ ddNTP Terminator Mix *10 mM*Water100 nmoles17205
ReadiUse™ dNTP Mix Set *10 mM PCR Grade*Water1 mL17258
ReadiScript™ RT Reverse Transcription Kit 50 Reactions60100



DNA sequencing with chain-terminating inhibitors
Single Nucleotide Polymorphism