dNTPs: Structure, Role & Applications

Deoxyribonucleotide Triphosphates dNTPs – A Comprehensive Overview

dNTPs  short for deoxynucleotide triphosphates are the building blocks of genetic information. dNTPs consist of a nitrogenous base, the desoxyribose sugar and a triphosphate bond to the hydroxy group of the next sugar linked to a base. The nitrogenous base can be the natural bases adenine, guanine, cytosine and thymidine or a modification such as 5-Ethynyluridine or similar. dNTPs can be synthesized enzymatically by various enzymes. However, for DNA forming reactions such as PCR, chemically produced dNTPs are usually used.

Understanding dNTPs – Building Blocks of DNA

During DNA replication (S Phase of cell cyclus), the dNTPs, dATP, dCTP, dGTP and dTTP, are used to synthesize a copy of DNA. Thereby, DNA polymerases catalyze the formation of a phosphodiester bond between the dNTPs. A dNTP is linked between the 3’ hydroxyl group of the last nucleotide in the strand of DNA and the first 5’ phosphate group of the dNTP.  The pyrophosphate bond of dNTPs is hydrolyzed in this process. The dNTP is converted to monophosphate by releasing a diphosphate group (pyrophosphate) in the process. The hydrolysis of the dNTP provides the energy to form the new phosphordiester bond.

The specific base-pairing of A:T and C:G through hydrogen bonds is essential for the characteristics of the DNA strands and helix formation. These hydrogen bonds lead to a stable double strand (dsDNA) to store the genetic information safely. However, it can also be easily separated into single-stranded DNA (ssDNA), e.g. by heating, to allow synthesis of new DNA as in PCR.

DNA Replication and Chain Elongation

DNA polymerases catalyze the formation of a phosphodiester bond. dNTPs are linked between the 3’ OH group of the last nucleotide in the growing DNA strand and the 5’ phosphate group of the next dNTP to be incorporated. This step releases two phosphate groups which provides the needed energy to form the new phosphor diester bond between the now linked dNTPs. Chain elongation varies depending on which strand is being replicated. The lead strand is replicated from dNTPs while the lagging strand is elongated discontinuously. Meaning short fragments are formed from dNTPs.

DNA Repair and Genetic Integrity

Many DNA polymerases have proofreading properties. Meaning they recognize the incorporation of wrong dNTPs, cleave them of and incorporate the right dNTP instead. During the process of DNA synthesis, DNA replication or repair gaps can be formed. For filling these gaps, dNTPs are incorporated to provide again a fully DNA strand. In base excision repair (BER) damaged bases are removed by generating a gap. With the correct dNTPs, DNA polymerase fills these gaps. Incorrectly incorporated dNTPs are removed by exonuclease activity of polymerases and replaced by the right dNTPs.

 

Importance of dNTPs in Genetic and Molecular Research

Modified deoxynucleotide triphosphates (dNTPs) are important tools in modern molecular biology and biochemistry due to their versatility and wide range of possible applications:

structure of 5-Ethynyl-dUTP (5-EdUTP)

Modified dNTPs as 5-Ethynyl-dUTP (EdUTP BCT-08) or its C8 variants (BCT-05 and BCT-06) can be used to to study DNA damage and their repair mechanisms. Such dNTPs can be incorporated into DNA strands by most polymerases, allowing for the enzymatic synthesis of single or multiple labeled DNA as well as chemically altered DNA. The incorporation of alkyne moieties enables post synthethic labeling with a wide variety of azide labelled dyes or tags by CuAAC. This process is used in multiple techniques such as PCR (Polymerase Chain Reaction) or DNA sequencing.

DNA Stability and Genetic Integrity

dNTPs are the building blocks of DNA and important for the replication and repair of DNA, especially across cellular divisions and generations. The correct use of dNTPs by DNA ensures the preservation of genetic information and DNA stability.

Genetic Research and Therapeutics

dNTPs and modified dNTPs are key molecules in modern biochemical research such as gene editing, next generation sequencing, personalized medicine or in studying genetic diseases. Gene technologies such as CRISPR-Cas9, base editing or prime editing are using dNTPs. This technology depends on the exact incorporation of modified or natural dNTPs to correct genetic mutations. For instance, prime editing benefits from the use of modified dNTPs to enhance the precision and efficiency of these edits. Next generation sequencing is a promising tool for personalised medicine. By using modified dNTPs into DNA or RNA sequence, genetic variations can be detected.

 

Applications of dNTPs in Research and Diagnostics

dNTPs are important units in various laboratory techniques and diagnostics like PCR, qPCR, Sanger and Next Generation Sequencing (NGS).

Essential Role in PCR and Sequencing

dNTPs are used in high important laboratory techniques as PCR and qPCR. Therefore, modified dNTPs are crucial ingredients which can be tagged with fluorescent dyes. These labelled dNTPs allowing real-time monitoring of DNA amplification. This is a particularly useful practice for diagnostics, such as the detection of viral infections, as was the case around the world during the Corona pandemic. For many NGS technologies modified dNTPs, especially clickable dNTPs are used to label them with fluorescents. This enables researcher to read sequences, base by base.

Genetic Testing and Molecular Experiments

There are several techniques like PCR or qPCR using modified dNTPs, e.g. for the detection of mutations. E.g. in prenatal genetic testing and the detection of genetic mutations and chromosomal abnormalities in fetal DNA. dNTPs and PCR is particularly useful for diagnosing Down syndrome, cystic fibrosis, and other genetic disorders.

 

Availability and Laboratory Use of dNTPs

Natural dNTPs and modified dNTPs are commonly used products in modern research. Therefore, a lot of procedures have been developed to synthesize dNTPs in high purity and yield. This leads to a wide offer of relatively cheap and pure unmodified and modified dNTPs for researchers.

Product Options for Research Applications

baseclick utilizes dNTPs in various innovative products and technologies, particularly in the fields of DNA labeling, sequencing and diagnostic. baseclick`s ClickTech PCR Modification kit contains all the necessary reagents for generating highly fluorescent labelled PCR products through a two-step method based on click chemistry. In these kits, dTTP is partially replaced by EdUTP depending on the desired labeling rate. EdUTP can be added to the native dNTP mixtures. This mixture is then used in standard PCR reactions with the optimized baseclick Ethynyl Polymerase. These kits include chemicals for performing multiple PCR and fluorescent labeling reactions, with different dyes available for various applications. ClickTech DNA FISH Kits use the same modified dNTPs to provide customers with all necessary chemicals to produce their own highly specific labeled DNA FISH probes.

dNTPs in baseclicks portfolio:

BCT-05 C8-Alkyne-dUTP

C8-Alkyne-dUTP can be used to introduce alkyne groups into DNA, enabling further functionalization through click chemistry. This approach allows for efficient tagging or conjugation, facilitating downstream applications like biomolecule tracking or immobilization.

BCT-06 C8-Alkyne-dCTP

Similar to C8-Alkyne-dUTP, C8-Alkyne-dCTP introduces alkyne groups into DNA for post-reaction with click chemistry, supporting applications such as DNA labeling and functionalized probe generation.

BCT-08 5-Ethynyl-dUTP

5-Ethynyl-dUTP facilitates DNA modification by incorporating ethynyl groups, ready for click chemistry applications, useful in creating labeled or functionalized DNA fragments.

BCT-21 Alkyne-dATP

Alkyne-dATP introduces an alkyne handle into DNA, which can then be labeled through click chemistry. This is ideal for creating modified DNA for research and diagnostic applications.

BCT-25 3’-Azido-2’3’-ddATP

3’-Azido-2’3’-ddATP supports DNA labeling with azides, allowing 3’-end site-specific modifications, beneficial in creating labeled DNA without complex production changes. 3’-Azido-2’3’-ddATP works as ddATP in Sanger sequence and leads after incorporation in PCR to an end of strand synthesis. This technique is used in ClickSeq’s NGS library preparation kits to prepare RNA or DNA libraries.

BCT-26 3’-Azido-2’3’-ddCTP

This azide-modified nucleotide supports DNA labeling with azides, allowing 3’-end site-specific modifications, beneficial in creating labeled DNA without complex production changes. 3’-Azido-2’3’-ddCTP works as ddATP in Sanger sequence and leads after incorporation in PCR to an end of strand synthesis. This technique is used in ClickSeq’s NGS library preparation kits to prepare RNA or DNA libraries.

BCT-27 3’-Azido-2’3’-ddGTP

Similarly, 3’-Azido-2’3’-ddGTP supports DNA labeling with azides, allowing 3’-end site-specific modifications, beneficial in creating labeled DNA without complex production changes. 3’-Azido-2’3’-ddGTP works as ddGTP in Sanger sequence and leads after incorporation in PCR to an end of strand synthesis. This technique is used in ClickSeq’s NGS library preparation kits to prepare RNA or DNA libraries. The dNTP is also used in the ClickTech Library Kit full-length mRNA_Seq V2.0 mRNA sequencing kit.

BCT-28 3’-Azido-2’3’-ddTTP

Like other azide-modified nucleotides, 3’-Azido-2’3’-ddTTP supports DNA labeling with azides, allowing 3’-end site-specific modifications, beneficial in creating labeled DNA without complex production changes. 3’-Azido-2’3’-ddTTP works as ddTTP in Sanger sequence and leads after incorporation in PCR to an end of strand synthesis. This technique is used in ClickSeq’s NGS library preparation kits to prepare RNA or DNA libraries.

BCT-34 2′-Azido-2′-dATP

2′-Azido-2′-dATP offers modular, chemoenzymatic DNA or RNA labeling, allowing flexible alkyne or DBCO-based bioconjugation at the sugar, ideal for creating labeled RNA without complex protocols.

 

Guidelines for Storage and Stability

Unmodified dNTPs and modified dNTPs are stable for long term storage at -20 °C. To increase stability it is useful to reduce freeze thaw cycles for dNTPs, therefore aliquotation of smaller samples is useful. dNTPs are stable in higher concentration, therefore it is useful to dilute just small amounts to lower concentrated working solutions of dNTPs. Keep dNTPs in dark light protected storage.