What Is dNTP?
Nucleoside triphosphate is a biomolecule involved in building nucleic acids (DNA and RNA). Structurally, they contain nitrogenous bases bound to a ribose sugar and three phosphates. And, based on the type of ribose sugar involved in their structure, such as ribose or deoxyribose, they are categorized as deoxyribonucleotide triphosphate (dNTP) or ribonucleotide triphosphate (NTP).
Deoxynucleotide triphosphate (dNTP) is a monomeric unit of DNA and formed by reducing ribonucleotides with the enzyme ribonucleotide reductase (RNR). It contains a nitrogen base, bound to deoxyribose sugar, and three phosphate groups attached to its 5’ carbon.
The nitrogenous group can either be a heterocyclic purine or pyrimidine structure, which supports the base-pairing interaction during DNA amplification. The specific pairing between these molecules allows nucleic acids to carry genetic information.
During DNA replication, a 5’ phosphate group of one nucleotide forms a phosphodiester bond with the 3′ carbon on another nucleotide via dehydration synthesis. Moreover, the addition of the new nucleotide always takes place at the 3’ end, implying the occurrence of DNA synthesis from the 5’ to 3’ end.
dNTP is of four types based on the nitrogenous base it contains in its structure:
A balance in dNTP pools in organisms is required to maintain and regulate genome integrity. Thus, any disturbances in the pool might lead to increased mutations, genome instability, and tumorigenesis.
Besides functioning as a building block of DNA molecules, these dNTPs have many applications in different life science and molecular biology labs, such as cloning, PCR reactions, and cDNA synthesis, especially for diagnostic and therapeutic purposes.
In this article, we will cover all the four types of deoxynucleoside triphosphate (dATP, dGTP, dTTP, and DCTP) and the laboratory applications of these dNTP components.
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Components of dNTP
Deoxyribonucleotide triphosphate (dNTP) is of four types based on the nitrogenous group (adenine, guanine, thymine, and cytosine) attached to the DNA building unit.
dTTP is one of the four nucleoside triphosphates required for DNA synthesis. It contains thymine as a nitrogenous base, which forms two hydrogen bonds with dATP (deoxyadenosine triphosphate) during DNA replication.
Thymine containing nucleoside triphosphate is primarily referred to as thymidine (instead of deoxythymidine) because of its zero roles in the transcription (mRNA synthesis) process.
The variety of functions performed by the biomolecule includes DNA replication, tRNA synthesis, and synchronizing cells at the G1/early S phase of the cell cycle. In labs, it’s used in a range of assays including DNA sequencing, DNA labeling, and cDNA synthesis.
dCTP is a nucleoside triphosphate containing cytosine as a nitrogenous base. During genome DNA replication, the nucleotide pairs with dGTP with three hydrogen bonds. The bonding between these nucleotides provides strength to the DNA structure.
The nucleotide is involved in the epigenetic modification of organisms’ genomes and DNA synthesis. In labs, it’s used in sequencing, library preparation for next-generation sequencing, and preparing a master mix for a variety of PCR reactions including qPCR, RT-PCR, and high-fidelity and long-range PCR.
dGTP is a nucleotide containing guanine as a nitrogenous base. It’s synthesized by dGMP via enzymatic synthesis and chemical phosphorylation.
It has a spectrum of roles in organisms, some of which include:
- DNA synthesis.
- Acting as an inhibitor of viral replication in HSV treatment.
- Acting as a substrate in nucleotide synthesis.
In molecular biology labs, it’s used in a variety of assays including qPCR, microarray, multiplex PCR, site-directed mutagenesis, genotyping, and molecular cloning.
dATP is one of the four building blocks of DNA. It has a molecular weight of 491.1816 g/mol and structurally consists of an adenine base, a deoxyribose sugar, and three phosphates.
The molecule has several roles including in vivo replication and transcription, acting as a metabolite in E.coli and mice, and the inhibition of human ribonucleotide reductase.
In labs, it’s used along with other reagents to carry out a spectrum of lab assays including PCR amplification, whole-genome sequencing, and DNA labeling and tailing.
Applications of dNTP
dNTP is a crucial reagent in molecular biology and other life sciences labs. It’s used to prepare the master mix required for a few experiments along with other reagents, which include Taq DNA polymerase, template DNA, and buffer.
Scientists either prepare the dNTP mix by themselves by adding individual dATP, dGTP, dCTP, and dTTP or purchase the dNTP mix from commercial providers. The DNTP concentration and the reagents used in the assays differ based on the experiment to be performed.
Before purchasing the commercial dNTP mix, ensure that the purity of the available product is >99% as confirmed by HPLC. Moreover, it should be free from any contaminants such as PCR inhibitors, DNases, RNases, and human and E.coli DNA.
The assays that were extensively performed in the labs and involve the use of the dNTP mix is given below.
Polymerase chain reaction (PCR) is a technique that utilizes DNA polymerase, such as Taq polymerase, to replicate the DNA segment into millions of copies.
In the experiment, dNTP is utilized to form new DNA strands using the template DNA during the extension phase of PCR.
The assay is used in many downstream applications including genetic mapping, forensics, disease screening, disease diagnostics, and genetic fingerprinting.
During DNA synthesis, dNTPs act as a unit to synthesize a complementary DNA strand from the template strand. The bases are provided to unzipped DNA to replicate the information.
dNTPs are utilized in a range of DNA labeling experiments, such as:
- 5’ end-labeling of DNA (using polynucleotide kinase and ATP-gamma-32P)
- 3’ end-labeling of DNA (using terminal transferase and deoxynucleotide triphosphate)
- Labeling a single nucleotide terminator
- Random priming extension labeling
- Nick translation labeling
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Deoxynucleotide triphosphate refers to the four nucleotides (dATP, dGTP, dTTP, and dCTP) that are involved in synthesizing DNA in organisms.
During replication, the nucleotides are added to the unzipped DNA for the formation of new complementary strands. The nucleotides on both the strands pair with each other through hydrogen bonding to form a double-strand helix.
dNTP is used in labs for a myriad of applications including DNA sequencing, DNA labeling, cDNA synthesis, and amplification of genomic DNA.
A high-quality and pure dNTP is required to ensure the integrity of the experiments. However, with that, advanced instruments and tools are also required to perform these high-throughput assays.
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