A Comprehensive Guide to ddNTP
The Definition of ddNTP?
ddNTP definition: Dideoxyribonucleoside triphosphates (ddNTPs) are artificial DNA nucleotides that are used in DNA sequencing according to Sanger sequencing technologies. Sanger sequencing, second generation sequencing and next-generation sequencing are used to identify DNA from victims or perpetrators, to detect genetic changes and variants such as gene insertions/deletions in patients, or to track mutational changes in viruses or others in the interest of public health. The ddNTPs are similar structured like the natural deoxyribonucleoside triphosphates (dNTPs). However, ddNTP structure is slightly different, the hydroxy group of ribose (sugar) at position 2‘ and 3’ is missing or replaced (modified ddNTP).
The chemical structure of ddNTP
ddNTP definition: The ddNTPs are similar structured like the natural deoxyribonucleoside triphosphates (dNTPs). However, ddNTP structure is different, the hydroxy group of ribose (sugar) at position 2‘ and 3’ are missing or being replaced (modified ddNTP). The addition of a small amount of a single ddNTP to the natural dNTP mixture in experimental setups such as PCR leads to chain termination. This results in PCR fragments of a DNA sequence of different lengths. The length of this fragments show where in DNA the base chosen to be partially replaced with the ddNTP is located. Using gel electrophoresis of separate experiments with all four ddNTPs, the different DNA bands can therefore provide a breakdown of the DNA sequence. Additional fluorescent labelling of ddNTPs at all four nucleobases (e.g. blue, green, red, yellow) enhances this technology and the ddNTP function by enabling multiplexing of the inserted base (A, G, C, T) by fluorescens detection of the corresponding color by a scanning laser/ fluorometer. This makes a single experiment sufficient for sequencing. Although the molecular weight and structure of ddNTPs differ from natural dNTPs, DNA polymerisation does not distinguish between them. Additional modifications such as fluorescent labels on the nucleobases of ddNTPs are also well accepted by polymerases, as demonstrated by companies such as Pacific Biosciences.
How ddNTP interrupts DNA replication
DNA sequencing is the detection and determination of the nucleotide sequence (A, G, C, T) in a DNA strand. The most common method of DNA sequencing is the Sanger sequencing method. Therefore ddNTPS are mixed to a natural dNTP mix during DNA synthesis. ddNTP definition/ ddNTP structure: ddNTP lack a 3′-hydroxyl group on the deoxyribose sugar ring but contain 5′-triphosphate and nucleobase. But what is the ddNTP function? The addition of a small amount of all four ddNTPs to the natural dNTP mix in experimental setups such as PCR leads to chain terminations. This produces PCR fragments of a DNA sequence with different lengths, as DNA polymerase cannot proceed without the essential 3′-OH group. That is the reason why the sanger method is also known as chain termination sequencing or the dideoxy method because the DNA elongation ceases.
The function of ddNTP in DNA Sequencing
The basic principles of Sanger sequencing: The controlled addition of a small amount of each of the four ddNTPs to the natural dNTP mixture during DNA synthesis causes a chain break. This produces DNA fragments of different lengths. Using gel electrophoresis, the different DNA bands can be precisely determined and the nucleotide sequences identified. It is not only Sanger sequencing that has gradually been automated. The development and integration into modern automated sequencing systems has led to various next-generation sequencing methods. The additional fluorescent labelling of ddNTPs at all four nucleobases (e.g. blue, green, red, yellow) improves the sequencing market and the ddNTP function by allowing multiplexing of the inserted base (A, G, C, T) by fluorescent detection of the corresponding colour by laser. The colour of the nucleobases ultimately indicates the sequence of the DNA strand.
Applications of ddNTP in Modern Research
Sanger sequencing, second generation sequencing and next-generation sequencing are used to identify DNA from victims or perpetrators, to detect genetic changes and variants such as gene insertions/deletions in patients, or to track mutational changes in viruses or others in the interest of public health. But in the most cases ddNTPs are used identifying disease genes and for pharmacogenetic studies via sequencing technologies. baseclick and ClickSeq are one of NGS kit producer which uses a new method and ddNTPs to study RNA, modify DNA, and make lab tests more sensitive and accurate. ClickSeq technology is a simple method for the synthesis of Next-Generation Sequencing (NGS) libraries and offer a Next Generation Sequencing (NGS) Library Prep Kit to finally sequence DNA or RNA. ClickSeq derives its names by using ‘Click-Chemistry‘ in the place of common ligation enzymes to ‘click-ligate’ nucleic acids to sequencing adaptors – an essential and often problematic step of Next Generation Sequencing (NGS) Library Prep Kits and in the synthesis of NGS cDNA libraries. ClickSeq using special ddNTP for artificial DNA creation and developing better DNA sequencing methods. The process takes advantage of the chain-terminating properties of 3′-azido-dideoxynucleotides (3’-azido-ddNTP), which are included in the initial in vitro reverse-transcription reactions uniformly required for RNAseq or DNAseq. The modified ddNTPs are stochastically incorporated into the nascent cDNA, yielding cDNA fragments blocked at their 3′ ends with azido groups. The 3′-azido-blocked cDNA fragments are ‘click-ligated’ onto alkyne-functionalized sequencing adaptors, which can subsequently be PCR-amplified to yield a sequencing-ready NGS library. Learn more about our 3’-azido-ddNTP and the advanced research applications and benefits of this specialised molecular biology technique using ClickSeq:
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.
CAS Number 1383937-03-8 (sodium salt)
Storage Conditions -20 °C
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 ddCTP 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.
CAS Number 92562-77-1 (free acid)
Storage Conditions -20 °C
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.
CAS Number 94059-38-8 (free acid)
Storage Conditions -20 °C
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.
CAS Number 149022-21-9 (sodium salt)
Storage Conditions -20 °C
ddNTP in high-throughput sequencing
Genetic sequencing is based on the efficient synthesis of DNA in which the four deoxyribonucleoside triphosphate (dNTP) substrates are partially replaced by dideoxyribonucleoside triphosphates (ddNTPs) carrying fluorescent labels. Sequencing by synthesis (SBS) is a widely used next-generation sequencing (NGS) technology worldwide, used by companies such as Illumina and Pacific Biosciences. To achieve base-by-base sequencing, four different spectrally separable fluorescent dyes are conjugated to each of the four ddNTPs. Size separation and sequence analysis are performed using capillary gel electrophoresis and a scanning laser. The color of each ddNTP-incorporated DNA band fragment is read by a fluorometer. This technology provides accurate data and high-throughput sequencing with improved accuracy, essential for analysing complex genomes. Optimal for large-scale projects.
Advantages and Challenges of Using ddNTP
For next-generation sequencing, ddNTPs are essential because this technique starts directly from genomic DNA or a cDNA library. The ddNTP-terminated DNA fragments are amplified and ligated to platform-specific oligonucleotide adapters, with the entire preparation process taking 90-240 minutes. The cost of sequencing one billion base pairs of DNA, measured in US$ is $5.83 (2022). Data adapted from National Human Genome Research Institute. The cost is divided into several items: DNA or RNA library preparation kits are required to produce DNA fragments of the nucleic acid of interest, ligation adapter kits are required to attach the correct primer for sequencing to the machine used, the machine itself plus chips and running material used to analyse the DNA/RNA libraries, software and a high-capacity computer to store the large amounts of data, and finally trained personnel to perform all the steps. There are technical challenges and limitations depending on the material and instrument used. There are manufacturers of low-cost sequencing instruments, but the accuracy is poor. There are manufacturers of DNAseq and RNAseq library preparation kits (using ddNTP), such as ClickSeq, with advantages such as no need for fragmentation or ligation, which improves accuracy and is less time-consuming. NGS instrument manufacturers are also scaling up their processes to millions of reactions in massively parallel fashion, enabling rapid sequencing of short DNA fragments over large parts of the genome or even entire genomes in a single run. In terms of throughput, the latest NGS instruments can generate hundreds of <350 megabases of data in a single sequencing run.