What is Deoxycytidine Triphosphate (dCTP)?

2′-deoxycytidine 5′-triphosphate (or dCTP) is a nucleoside triphosphate (a nucleotide), containing cytosine as its pyrimidine base. It also has deoxyribose sugar attached to three phosphate units. Its chemical formula is C₉H₁₆N₃O₁₃P₃ and has a molecular weight of 467.1569 g/mol. The cas number of the compound is 2056-98-6—this number is given by the Chemical Abstracts Service (CAS) to refer directly to the compound’s information.

In the double-stranded DNA, dCTP is paired with dGTP, forming three hydrogen bonds, whereas dATP and dTTP make a pair by forming two hydrogen bonds. So, the GC content in the strand contributes maximum to the stability of the structure and determines its physical properties, such as its melting temperature.

Some other derivatives of cytosine (such as 5-methyl-cytosines that is formed after methylation and dUTP that result after bisulfite treatment), also have essential functions to perform in organisms.

Bisulfite treatments and the use of methylation-sensitive restriction enzymes (with or without methylation treatment) are two methods that are utilized to detect the methylation of dCTP in genomic DNA.

This article provides a complete overview of what dCTP is and its uses in labs and the body of other organisms. Furthermore, it covers its structural representations and its working mechanisms.

dCTP Use Cases & Functions

dCTP (including other dNTPs, such as dATP, dGTP, dCTP, and dGTP) has several uses inside the body and molecular biology labs. Essentially, they help determine the working mechanisms of organisms’ metabolic functions. Furthermore, they are also used to study the evolution of organisms or the categorization of any new species to a specific family (through DNA sequencing).

Some additional uses and functions of dCTP are explained below:

• In labs, dCTP (and other dNTPs) is used in assays like sequencing, cloning, cDNA synthesis, and library preparation for next-generation sequencing.
• A concentration of approx 100 mM solution (in water) of dCTP is prepared for in vitro PCR, reverse transcription, RT-PCR, real-time PCR, high-fidelity, and DNA labeling reactions (in addition to other reagents and consumables, including template DNA, gene-specific primer pair, dNTP mix, tris buffer, water, thermal block cycler, and PCR tubes).
• Dye dCTP is used for microarrays, in situ hybridization, and blotting applications to produce labeled DNA probes.
• In the body of organisms, it’s used to synthesize DNA (deoxyribose nucleic acid).
• It’s also involved as a metabolite in several biological processes, including dihydropyrimidinase deficiency, beta ureidopropionase deficiency, pyrimidine metabolism, UMP synthase deficiency, and mitochondrial neurogastrointestinal encephalopathy.

Cytosine modified at carbon five is a significant epigenetic modification, having an array of roles. Some of its functions in organisms are:

• The modified compound acts as a substrate for the family of Ten-Eleven Translocation (TET) enzymes and related oxidation pathways.
• In addition to its ability to generate completely methylated, cytosine-substituted DNA, 5-methyl-dCTP can also be employed for a wide array of biochemical and cellular functions.
• A solution of 5-methyl-dCTP is prepared in 10 mM solution in Milli-Q™ water, and the absorbance is taken at 260 nm to measure the nucleotide concentration.

The commercially available dCTP, used in most lab workflows, can be stored at -20 degrees celsius. It’s also highly recommended to refer to the material and safety data sheet (MSDS or SDS) to learn about the quality of the dCTP nucleotide that you are purchasing for your experiment.

Moreover, make sure that the datasheet highlights these four qualities:

• High stability of the compound.
• Purity equals to or is more than 99%.
• Is tested using HPLC.
• Free of human and E. coli DNA.

How Does dCTP Work?

dCTP contains three phosphates joined together by phosphoanhydride bonds. These high-energy anhydride bonds release energy when undergoing hydrolysis (a reaction in which water is utilized to break one or more chemical bonds between the molecules).

During DNA replication, the energy released due to the breakage of high-energy bonds is utilized by the enzyme, DNA polymerase, to add dCTP (and other dNTPs) into the newly synthesized DNA strand.

(DNA)n + dCTP ↔ (DNA)n-C + PPi

During the DNA synthesis process:

• The pyrophosphate bond (PPi, in which two phosphorus atoms are arranged as P–O–P) present in dCTP is cleaved off.
• The breakage of bonds leads to the formation of nucleoside monophosphate.
• The nucleoside monophosphate is incorporated at the 3’ end of the newly formed DNA.

How is dCTP incorporated into DNA, along with the other dNTPs?

dCTP is one among four dNTP (deoxynucleotide triphosphates) that form the DNA structure (along with ribose sugar and triphosphate).

• DNA forms in the 5’ to 3’ direction.
• All the incoming bases are added to the 3’ end of the growing chain.
• The 3’ end of the DNA contains a hydroxyl group, and upon encountering an incoming nucleoside triphosphate, this hydroxyl group attacks the alpha phosphate group.
• A pyrophosphate molecule is ejected from the complex as a result of the 3′-OH replacing the beta and gamma phosphates.
• Phosphodiester bonds are formed between the growing daughter strand and the next nucleotide.
• The reaction then repeats, and in the same way, subsequent nitrogenous bases are added to the growing strand.

dCTP Structure

dCTP is composed of a pyrimidine cytosine base, a pentose sugar (deoxyribose), and three phosphate groups.

Its preferred IUPAC name—a name given to compounds by IUPAC based on their structure and chemical composition is:

[[(2R,3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxy oxolan-2-yl]methoxy-hydroxy phosphoryl] phosphono hydrogen phosphate

Figure: A 2-dimensional structural representation of deoxycytidine triphosphate (dCTP).

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CTP (cytidine triphosphate) should not be confused with dCTP (deoxycytidine triphosphate). Cytidine triphosphate contains ribose with a hydroxyl group at its 2’ position, whereas deoxycytidine triphosphate contains deoxyribose sugars (which has only a hydrogen atom at its 2’position, instead of the hydroxyl group). CTP is used in the formation of RNA, while dCTP is used in DNA.

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Deoxycytidine triphosphate (dCTP) is a nucleoside base composed of cytosine, deoxyribose sugars, and three phosphate groups. It’s involved in forming DNA structures, along with other nitrogenous bases that include adenine, guanine, and thymine.

The molecule also acts as a metabolite in several metabolic pathways and is involved in certain diseases. It’s also utilized in labs in a spectrum of in vitro molecular biology workflows, including PCR, DNA sequencing, and determination of nucleotide concentration.

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