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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.
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
An illustration of Sanger sequencing by capillary electrophoresis (figure made in BioRender).
During Sanger Sequencing:
- The unknown DNA sequence is combined with dNTPs, DNA polymerase and a (sometimes radioactive-linked) primer.
- 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.
- The mix is heated to denature the template DNA, then cooled to allow for primer binding.
- 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.
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Products
Table 2. Available MagaDye™ fluorescent ddNTPs for Sanger sequencing.
| Product Name ▲ ▼ | Nucleotide ▲ ▼ | Ex (nm) ▲ ▼ | Em (nm) ▲ ▼ | Abs(nm) ▲ ▼ | Unit Size ▲ ▼ | Cat No. ▲ ▼ |
| MagaDye™ 535-ddGTP | Guanine | 503 nm | 536 nm | 503 nm | 5 nmoles | 17063 |
| MagaDye™ 535-ddGTP | Guanine | 503 nm | 536 nm | 503 nm | 50 nmoles | 17067 |
| MagaDye™ 561-ddATP | Adenine | 498 nm | 561 nm | 498 nm | 5 nmoles | 17062 |
| MagaDye™ 561-ddATP | Adenine | 498 nm | 561 nm | 498 nm | 50 nmoles | 17066 |
| MagaDye™ 588-ddTTP | Thymine | 498 nm | 588 nm | 498 nm | 5 nmoles | 17061 |
| MagaDye™ 588-ddTTP | Thymine | 498 nm | 588 nm | 498 nm | 50 nmoles | 17065 |
| MagaDye™ 613-ddCTP | Cytosine | 498 nm | 614 nm | 498 nm | 5 nmoles | 17060 |
| MagaDye™ 613-ddCTP | Cytosine | 498 nm | 614 nm | 498 nm | 50 nmoles | 17064 |
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 µmoles | 17070 |
| Aminopropargyl ddTTP [5-Propargylamino-2',3'-dideoxyuridine-5'-triphosphate] | 10 µmoles | 17072 |
| Aminopropargyl ddATP [7-Deaza-7-Propargylamino-2',3'-dideoxyadenosine-5'-triphosphate] | 10 µmoles | 17074 |
| Aminopropargyl ddGTP [7-Deaza-7-Propargylamino-2',3'-dideoxyguanosine-5'-triphosphate] | 10 µmoles | 17076 |
| 2-Aminoethoxypropargyl ddCTP | 1 µmoles | 17080 |
| 2-Aminoethoxypropargyl ddTTP | 1 µmoles | 17082 |
| 2-Aminoethoxypropargyl ddATP | 1 µmoles | 17084 |
| 2-Aminoethoxypropargyl ddGTP | 1 µmoles | 17086 |
| 7-Deaza-7-Propargylamino-3'-azidomethyl-dATP | 50 nmoles | 17090 |
| 5-Propargylamino-3'-azidomethyl-dCTP | 50 nmoles | 17091 |
Table 4. Deoxynucleotides (dNTPs) for use in PCR, real-time PCR, and reverse transcription PCR