dUTP: Application & Overview

dUTP: Overview

Deoxynucleotide triphosphates (dNTPs) are the building blocks of the DNA molecules, the genetic material of most organisms. It includes dATP, dTTP, dGTP, dCTP, and dUTP. During DNA replication, these nucleotides lose two phosphate groups to get incorporated into the amplifying chain of DNA.

dUTP is deoxyuridine phosphate that is a close structural congener of dTTP (deoxythymidine triphosphate). The nucleotide can be easily incorporated into organisms’ genomes by pairing with dATP during replication in the place of dTTP. The reaction is known as uracilation, which leads to non-mutagenic dU/A base pairs.

Structurally dUTP consists of a deoxyribose sugar, three phosphates, and a pyrimidine base—uracil.  The IUPAC name of 2′-Deoxyuridine 5′-triphosphate is [[(2R,3S,5R)-5-(2,4-dioxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate. 

Figure: The structure of the deoxyuridine triphosphate (dUTP).

The other form of dUTP is UTP which is involved in the formation of RNA during transcription. Unlike dUTP, UTP contains a ribose sugar, which has a hydroxyl (OH) group at position 2 of the pentose structure.

Commercially available dUTP reagents (supplied as a sodium salt in ultrapure water at pH 7.5) are used in various in vitro workflows, such as cDNA synthesis, cloning, sequencing, and genotyping. In this article, we will cover more about dUTP’s features and lab applications.

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How Does dUTP Work?

dUTP is incorporated into DNA during replication by pairing with dATP replacing dTTP. Inside an organism this is an abnormal pairing and needs to be removed. The molecule has more applications in lab workflows compared to organisms’ bodies. 

Commercially, the dUTP reagent is available as a kit, standalone enzyme, and also as a master mix, which can be used based on the application. However, before purchasing such lab-grade reagents, it’s essential to ensure their quality by referring to the certificate of analysis, safety data sheet (SDS), and citations showing product use. 

Here are some features that the manufacturers should strictly emphasize about their reagent: 

  • Greater than 99% purity as determined by HPLC
  • Highly stable
  • Free of human and E. coli DNA
  • Free from DNA and PCR inhibitor

By using dUTP instead of or in conjunction with dTTP, the PCR product generated during the reaction becomes a suitable substrate for the enzyme Uracil DNA Glycosylase (UDGase). The enzyme enables researchers to completely eliminate the contamination of DNA from previous PCR reactions before proceeding with the current amplification.

dUTP Pyrophosphatases

During DNA replication, dUTP pyrophosphatase hydrolyzes dUTP to dUMP and pyrophosphate (PPi), which prevents the incorporation of uracil into DNA. The enzyme is essential for accurate DNA replication and repair mechanisms in normal organisms. 

The reaction carried by dUTP Pyrophosphatases reduces dUTP levels in organisms and offers more dUMP for the biosynthesis of dTTP, which is required for normal DNA synthesis. The cellular dTTP:dUTP ratio must be high in order to prevent uracil incorporation into DNA, which results in DNA strand breaks and cell death.

What is dUTP Used For?

dUTP has a spectrum of applications in molecular biology lab workflows, which include:

  • Standard or hot-start PCR
  • Long-range and high-fidelity PCR
  • qPCR assay
  • cDNA synthesis and RT-PCR
  • Real-time PCR and RT-PCR with Taq DNA polymerase
  • Sequencing
  • DNA labeling
  • Nucleic Acid (DNA and RNA) Labeling & Oligo SynthesisPCR
  • Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase.
  • Primer extension.
  • Label secondary antibodies

It should be noted that dUTP cannot be used in PCR with proofreading enzymes standalone or enzyme mixes that include proofreading enzymes.

Some other forms of dUTP used in PCR assays include:

  • Amino-11-dUTP: It’s used for PCR, reverse transcription, microarray labeling, and nick translation. Nucleotide incorporation is carried out by Taq polymerase, reverse transcriptases, and Klenow fragments.
  • 5-Aminoallyl-dUTP: It can be enzymatically incorporated into DNA. It results in the formation of amine-containing DNA that can be easily labeled with biotin, fluorescent dye, or other haptens using classical peptide coupling techniques. 

dTTP Replacement

dUTP is frequently used in molecular biology lab experiments, such as RT-PCR and PCR assays, as a replacement for dTTP to avoid carryover from previous amplifications. In order to prevent false positives resulting due to the addition of uracil, the enzyme uracil-N-glycosylase is added to the PCR premix. To ensure that dUTP is free of nuclease activity, each lot of dUTP should be tested for specific DNA amplification.

Preamplification

Preamplification is necessary when analyzing rare DNA and RNA molecules in small sample sizes, such as single cells and liquid biopsies. It makes downstream analyses particularly susceptible to contamination from polymerase chain reactions (PCRs). And to eliminate carry-over contamination, uracil-DNA glycosylase (UNG) is used before initiating the PCR reaction.

What Industries Use dUTP?  

Because of the diverse applications of dUTP in lab workflows, such as sequencing, PCR assay, DNA and antibody labeling, and cDNA synthesis, the molecule is widely used in medical, clinical, biotech, and pharma industries.

Pharmaceutical

dUTP is widely used in Pharma industries to quantify nucleic acid levels in cells or tissue biopsies. Further, it’s also used in detecting microbial forms and quantifying pathogen load during a disease condition. The other form of dUTP, such as dUMP, has an essential role in  DNA replication and repair.

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dUTP or deoxynucleotide triphosphate is a nucleotide used in vitro applications. The molecule consists of a deoxyribose sugar, three phosphates, and a uracil base. In normal organisms, the molecule pairs with dATP, replacing dTTP, during DNA replication. This abnormal base pairing needs to be excised for original DNA synthesis. 

dUTP is extensively exploited in labs in a range of workflows, such as sequencing, hot-start PCR, RT-qPCR, DNA labeling, and cDNA synthesis. It’s mainly to eliminate carry-over contamination coming from previous PCR reactions to the current amplification process.

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 Excedr can help your lab get the technology and equipment it deserves. With the right equipment, you can accelerate your R&D and achieve milestones faster! Get your customized leasing solution now.