ddATP: Understanding its role & applications
ddATP dideoxyadenosine triphosphate is a modified didesoxyribonucleosid-triphosphate that is used especially in sequencing technologies such as Sanger sequencing. The ddATP are similar structured like the natural deoxyadenosine triphosphates (dATPs) but with a slightly difference. Polymerization with further triphosphates is not possible because the hydroxy group of ribose (sugar) at position 2‘ and 3’ is missing or replaced.
What is ddATP?
The structure of dideoxyadenosine triphosphate
Dideoxyadenosine triphosphate (ddATP) is a nucleoside triphosphate (adenosine triphosphate – ATP) modified in its deoxyribose sugar moiety. In ddATP, the deoxyribose sugar lacks two hydroxy groups at the 2′ and 3′ positions of the sugar ring in comparison with the regular deoxyribose in dATP, hence the name “dideoxy”. Hydrogen atoms replace the 2′ and 3′ hydroxyl (-OH) groups. The absence of the 3′-hydroxyl group in ddATP prevents the formation of the phosphodiester bond between complementary nucleotides. This means that the incorporation of ddATP into a growing DNA strand blocks further nucleotide addition and stops the elongation process. In the Sanger method of DNA sequencing, ddATP is used as a chain terminator to control DNA fragmentation during sequencing.
Role of ddATP in DNA sequencing
ddATP is an important building block in Sanger DNA sequencing. Dideoxyribonucleosid-triphosphates are used as chain terminators in this technology. Because ddATP lacks a 3′ hydroxyl group (OH) on its deoxyribose sugar, it prevents the addition of further nucleotides to the growing DNA strand by DNA polymerase and stops the DNA chain elongation. This specificity enables the generation of DNA fragments of different lengths, which are necessary for sequencing. ddATP is incorporated at random positions during DNA synthesis. The resulting DNA fragments contain the ddATP at the ends, allowing the DNA nucleotide sequence to be determined by analyzing the length of the fragment. DNA fragments are usually analyzed by gel electrophoresis or fluorescence detection to determine the original DNA sequence template.
Importance of ddATP in genetic & molecular research
ddATP is used in many genetic research because it enables the controlled termination of DNA synthesis. Thus, Sanger sequencing (also known as chain termination sequencing) is used to identify mutations and changes in DNA, such as single nucleotide insertions, and deletions. The process begins with the denaturation of the double-stranded DNA followed by the annealing of the single-stranded DNA to oligonucleotide primers and elongated by DNA polymerase using a mixture of regular nucleotides dNTPs and a small amount of chain-terminating ddNTP, including ddATP. Since dNTPs and ddNTPs have an equal chance to be incorporated to the strand, each sequence is terminated at a different length. Further, ddATP and other ddNTPs can be labelled with a fluorescent marker or tags. When a labelled ddNTP is incorporated into the sequence, the base is fluorescent. Further elongation of the DNA is stopped, resulting in fragments of different lengths corresponding to specific positions in the DNA sequence. The fragments are analyzed by electrophoresis and the sequence is determined from the unique fluorescent signals emitted by each ddNTP, including labelled ddATP. Mutations can be identified by comparing the obtained sequence with a reference genome. This technique can also be used to determine the exact position of a gene or genetic marker on a chromosome, or to discover disease-related genes. Sanger sequencing based on the use of ddNTPs, including ddATP, helps researchers in biology and medicine to decode the genetic code and identify genetic variations, perform high-throughput sequencing (HTS) projects, contribute to understanding of genetic diseases, personalized medicine, and gene therapy. This method enables personalized medicine, where sequencing technologies using ddATP identify an individual’s genetic profile and specific disease related genetic mutations, enabling the development of targeted therapies and gene-editing strategies.
In addition to NGS kits using ddNTPs including ddATP, baseclick also offers high quality azide–modified ddATP nucleotides. This dideoxyadenosine triphosphate, which baseclick offer in high purity, is an azide-modified nucleotide that closely resembles a natural nucleotide and enables chemoenzymatic labeling of ssDNA or RNA. This product enables the precision of genetic research and accurate gene mapping using chain termination sequencing and click chemistry for analysis.
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
ddATP in laboratory setting
ddATP is available in different solid forms available as free acid, dry powder or dissolved in buffers with different concentrations. Solid ddATP is typically stabilized with counter ions, sodium or potassium, lithium salt solutions. ddATP can also be supplied in chemically modified forms to increase stability or its application such as click modification.
Concentration of ddATP:
High concentration ddATP: 200 µM – 100 mM commonly used for chain termination in Sanger sequencing and other sequencing methods. Standard concentration ddATP: 10 mM is suitable for a wide range of molecular biology methods, for example, PCR, qPCR, nick translation, cDNA synthesis, TdT-tailing reactions. baseclick offers the azide modified 3’-Azido-2’3’-ddATP in two versions as 100mM solutions in two different sizes, 1 µmol (BCT-25-S) and 5 µmol (BCT-25-L).
Storage & handling guidelines
ddATP is stable and effective for laboratory experiments if these best practices and ideal storage conditions are followed. However, dideoxyadenosine triphosphate as a chemical product must be stored under appropriate conditions to ensure its stability and effectiveness. For long-term storage, ddATP is stored at -20°C, which helps maintain its stability and prevents degradation. ddATP can be stored at +4°C for short-term handling up to one week without significant loss of activity. To avoid freeze-thaw cycles and minimize degradation ddATP store in small aliquots. Store ddATP in dark vials or boxes to protect it from light, that can cause degradation. A low moisture environment or the use of desiccants is required during solid ddATP storage to prevent exposure and contamination. Expiry date should always be checked and ddATP should be tested for activity periodically.
Future of dideoxyadenosine triphosphate in biotechnology
ddATP has traditionally been used as a chain terminator in Sanger sequencing, but it is also being used in new and emerging technologies. It is becoming increasingly important for tracking biodiversity and monitoring ecosystem changes. It can also be used in agricultural biotechnology to identify genes for improving yield, and environmental resilience in cultivated plants. However, ddATP is most commonly used to identify individual genetic variants for diagnosis or detection of rare mutations in cancer. The future role of ddATP in DNA sequencing in:
High-throughput sequencing: NGS technologies achieve high accuracy and throughput influenced by chain termination principles. ddATP remains essential in hybrid systems that merge the precision of Sanger sequencing with the high throughput of next-generation technologies, particularly for validating clinical mutations.
Long-read sequencing: Platforms such as PacBio and Oxford Nanopore offer detailed genomic analysis, and chemically modified ddATP can be used to enhance signaling and controlled termination.
Single-Molecule Real-Time (SMRT) Sequencing: Using PacBio’s SMRT technology researchers can watch the synthesis of DNA in real time, offering more accurate genetic sequences. In this context, ddATP enables precise control over DNA strand termination, which can be in programmable molecular systems and data encoding.