ddTTPαS , CAS No.158705-77-2

CAS: 158705-77-2 Cat. No.: D1446957 Formula: C₁₀H₁₇N₂O₁₂P₃S Peso molecolare: 482.23

Storage

Store at -20°C

Shipped In

Ice chest + Ice pads

Panoramica

ddTTPαS is a dideoxynucleotide that can be used for DNA sequencing.

Specifications

Condizioni di conservazione di stoccaggio

Store at -20°C

Spedito in

Ice chest + Ice pads

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Nomi e identificatori

Peso molecolare

482.23

Application Protocols

Example protocol 1 (literature-style, for primer extension termination with ddTTPαS):

• Prepare reaction (20 µL): 1× polymerase buffer, 1.5–2.5 mM MgCl2, 200 µM each dATP/dCTP/dGTP, 50 µM dTTP, ddTTPαS at 5–20 µM (titrate), 0.5 µM primer annealed to template, and 1–5 U exonuclease-deficient DNA polymerase. Adjust water to volume.
• Incubate at the enzyme’s optimal temperature for 5–30 min.
• Quench with 10 mM EDTA and formamide loading dye if analyzing by denaturing PAGE; heat 95°C for 2–3 min and load.
• Expect termination at template A positions; optimize ddTTPαS:dTTP ratio for band intensity spacing.

Example protocol 2 (capillary sequencing spike-in):

• Set up standard dye-terminator sequencing mix; replace or supplement ddTTP with ddTTPαS at 2–10 µM to enhance 3′-end nuclease resistance of terminated fragments (pilot titration required).
• Proceed with thermal cycling per kit instructions; purify and analyze as usual.

Good practices:

• Prepare fresh working aliquots in nuclease-free water or low-salt Tris buffer (pH ~7.5–8.5).
• Include control reactions with ddTTP (non-αS) to benchmark incorporation efficiency.

All values above are general literature guidance only and may require adjustment for your specific enzyme/template system.

Biological Roles

Item-specific biological roles are not applicable in vivo; this is a synthetic nucleotide analog for research. No clinical or therapeutic claims are made.

General/literature context:

• Structural motif: dideoxynucleotides mimic natural deoxynucleotides but lack the 3′-OH, causing termination of DNA chain elongation when incorporated by DNA polymerases.
• α-Thiotriphosphate modification: Upon incorporation, yields a phosphorothioate internucleotide bond, which is more resistant to many nucleases and exhibits altered stereochemical interactions with proteins and metal ions.
• Enzymology: Used to probe polymerase active-site chemistry, metal-ion coordination at the α-phosphate, and to modulate exonuclease proofreading susceptibility.
• Biophysical implications: Phosphorothioate linkages can perturb local charge distribution and coordination geometry, enabling mechanistic studies of catalysis and DNA–protein interactions.

Use strictly in vitro or in cell-free systems unless part of controlled research protocols; not intended for biological exposure or therapeutic applications.

Buffer Applications

This product is not a buffering agent and is not used to prepare buffer systems. It is used as a reagent within pre-formulated polymerase buffers.

Practical note:

• Prepare and use ddTTPαS in the buffer supplied or recommended for the chosen DNA polymerase (typically Tris-based with KCl and MgCl2). Adjust only the nucleotide and metal ion concentrations required for your enzymatic assay.

Green Alternatives

This product is a specialized aqueous biochemical reagent rather than a process solvent. Green chemistry considerations focus on minimizing hazardous components and waste in enzymatic workflows.

Greener practice opportunities (general):

• Use aqueous buffers exclusively; avoid organic co-solvents.
• Replace radioisotope-based detection with fluorescent or mass-based detection to reduce radiological waste.
• Employ thermostable enzymes that enable lower reagent loads and higher specificity, reducing repeat reactions.
• Scale reactions to the minimal volume necessary to achieve analytical goals, reducing nucleotide and plastic waste.

Comparison of options (informational):

| Option | Stability to nucleases | Incorporation efficiency | Waste considerations | |---|---|---|---| | ddTTP (standard) | Lower at 3′ terminus vs αS | Higher | Similar aqueous waste | | ddTTPαS | Higher (phosphorothioate) | Lower to moderate (enzyme-dependent) | Similar aqueous waste; fewer repeats may offset usage |

Trade-offs:

• ddTTPαS may reduce the need for nuclease inhibitors or repeated runs due to improved stability of products, potentially lowering total reagent consumption. Conversely, slightly reduced incorporation efficiency can require optimization to avoid excess reagent use.

Pharmaceutical Uses

No pharmaceutical or clinical uses are claimed or supported for this product. It is intended solely as a research reagent.

Formulation/excipient status (general):

• ddNTP analogs like ddTTPαS are not used as excipients. They are specialty substrates for in vitro enzymatic reactions (e.g., sequencing, footprinting, and mechanistic biochemistry).

Regulatory note:

• Not a compendial (USP/EP) excipient; no drug product use is indicated. For regulated applications, qualify the material per your quality system, and consult the CoA/SDS.

Physical Properties

Item-specific properties (from Product Data):

• Appearance: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.

General/literature expectations for ddNTPαS materials (informational, not specifications):

• Physical state: typically supplied as a lyophilized solid or aqueous solution of the sodium or lithium salt.
• Solubility: highly soluble in water and aqueous buffers; negligible solubility in organic solvents.
• Acid/base character: polyacidic (multiple phosphate pKa values); solutions are commonly adjusted to pH ~7.0–8.5 for stability and enzyme compatibility.
• Hygroscopicity: can be hygroscopic; solutions are sensitive to hydrolysis at elevated temperature or extreme pH.
• UV absorbance: thymine chromophore shows λmax near 260 nm (literature; extinction coefficient depends on ionic state and solvent).
• Volatility: non-volatile, decomposes before boiling.

Handling implications (general):

• Prepare and store in nuclease-free, low-metal ion containers to minimize degradation.
• Avoid repeated freeze–thaw; aliquot solutions for long-term use.

Quality and Grades

Item-specific grade/purity: Not specified for this item; refer to CoA/Spec Sheet.

Interpretation and typical expectations for nucleotide reagents (general guidance):

• Research-grade ddNTPαS reagents are commonly qualified for molecular biology applications (e.g., primer extension, Sanger/enzymatic incorporations). Key quality attributes often include:
  • High chemical purity and correct counterion content.
  • Low residual solvents and salts; low nuclease and phosphatase activity (RNase/DNase-free).
  • Verified identity by HPLC/LC–MS and NMR (31P can confirm α-thio substitution and diastereomer ratio; provided on CoA in many cases).
• UV transparency and baseline cleanliness matter for accurate quantification at 260 nm; suppliers may provide an A260 value and extinction coefficient for concentration calculations (consult CoA).

Stabilizers/buffers:

• Some ddNTPαS products are supplied in Tris or sodium bicarbonate at pH ~7–8 to maintain stability. If stabilizers are present, they will be listed on the CoA; none are specified here.

What to check on receipt:

• Lot-specific concentration (if solution), counterion, pH, diastereomeric composition (Rp/Sp) if reported, and recommended working concentration range for your application.

Reaction and Applications

Core use-cases (literature/general):

• Chain-termination sequencing and primer extension: ddTTPαS acts as a thymine-specific chain terminator due to the 3′-H (no 3′-OH). The α-thio modification yields a 3′→5′ phosphorothioate linkage at the site of incorporation, enhancing nuclease resistance of the newly formed terminus.
• Enzymatic incorporation to introduce a terminal phosphorothioate: useful for mapping, footprinting, or generating nuclease-resistant 3′ ends in DNA constructs.
• Polymerase selectivity studies: evaluate discrimination between normal dNTPs, ddNTPs, and α-thio analogs across polymerase variants (thermostable, high-fidelity, or exonuclease-deficient enzymes).

Practical guidance:

• Enzymes: Many DNA polymerases can incorporate ddNTPαS, but efficiency varies. Exonuclease-deficient Taq/Tth/T7 variants and certain reverse transcriptases may show improved tolerance. Pilot titrations are recommended.
• Ratios: Using micromolar ddTTPαS against 10–200 µM native dNTPs is common in termination/competition assays; optimize for desired termination frequency (literature guidance).
• Metal ions: Mg2+ at 1.5–3 mM is typical for polymerase buffers; Mn2+ can increase incorporation of non-canonical analogs but may reduce fidelity.
• Temperature/pH: Follow enzyme manufacturer recommendations (e.g., 50–74°C for thermostable polymerases; pH 8.0–9.0 at room temperature for Tris buffers), as incorporation kinetics and stability depend on these parameters.
• Quenching/analysis: Products are typically analyzed by capillary electrophoresis, PAGE, or HPLC; phosphorothioate linkages confer altered cleavage patterns with nucleases and may shift mobility slightly.

Notes:

• Expect lower incorporation rates than ddTTP; αS substitution can further reduce efficiency depending on the enzyme and Rp/Sp isomer.
• For radiolabel-free workflows, combine with fluorescent primers or labeled dNTPs for detection.

Reaction Conditions

The following are general literature guidelines for enzymatic use; they are not product specifications. Optimize for your enzyme and template.

Typical primer-extension/Sanger-style conditions:

• Buffer: polymerase-supplied buffer (e.g., 10–20 mM Tris-HCl, pH 8.0–9.0 at 25°C), 50 mM KCl (or as recommended), 1.5–3.0 mM MgCl2.
• Nucleotides: 10–200 µM canonical dNTPs; ddTTPαS at 1–50 µM depending on desired termination frequency and enzyme tolerance.
• Enzyme: exonuclease-deficient variants often improve incorporation of dideoxy/αS analogs.
• Temperature: as recommended for the polymerase (e.g., 60–74°C for thermostable polymerases; 37°C for mesophilic enzymes like Klenow exo−).
• Time: 5–60 minutes for primer extension, or thermocycling programs for PCR-like protocols.
• Additives: Mn2+ (0.1–0.5 mM) may enhance incorporation of non-canonical substrates at the cost of fidelity (use cautiously). Betaine/DMSO can assist with GC-rich templates but should be minimized to avoid affecting nucleotide stability.

Workup and analysis:

• Quench by EDTA addition and cooling; analyze on denaturing PAGE or capillary electrophoresis. Phosphorothioate-terminated products display enhanced resistance to 3′→5′ exonucleases and altered cleavage by certain endonucleases.

Notes:

• Rp/Sp isomer effects: polymerases can display stereoselectivity at the α-phosphate; mixed isomer preparations may show averaged kinetics.
• Always include appropriate controls (no-ddNTP, ddTTP control) to calibrate termination efficiency.

Safety and Handling

GHS/SDS information (item-specific):

• Signal Word: Not specified for this item; refer to SDS.
• H-Statements: Not specified for this item; refer to SDS.
• GHS Classification: Not specified for this item; refer to SDS.
• Pictograms: Not specified for this item; refer to SDS.

General laboratory safety guidance (informational):

• PPE: wear lab coat, safety glasses, and appropriate gloves (e.g., nitrile). Handle powders/solutions in a clean area to avoid contamination with nucleases or metal ions.
• Avoid inhalation of powders and contact with eyes/skin. Wash thoroughly after handling.
• Storage incompatibilities: avoid strong acids/bases (can accelerate phosphate hydrolysis), strong oxidizers, and divalent metal contamination (can catalyze degradation). Store away from light to protect the nucleobase chromophore.
• First aid overview: rinse skin/eyes with water for several minutes if exposed; remove contaminated clothing; if inhaled, move to fresh air; if ingested, rinse mouth. Seek medical attention as per institutional protocol.
• Environmental precautions: prevent release to drains in bulk; collect waste according to institutional chemical waste procedures.

Special risks (general for nucleotides):

• Aqueous solutions may be biologically benign but can support microbial growth if contaminated; use sterile, nuclease-free techniques.
• Do not autoclave nucleotide solutions; heat can cause decomposition. Filter-sterilize if sterility is required.

Always defer to the product’s SDS and local EH&S guidance for authoritative safety information.

Solvent Selection

This product is a polar nucleotide reagent, not used as a bulk solvent. Solvent selection here refers to choosing an appropriate medium for dissolution and enzymatic reactions.

Recommended media (general/literature):

• Primary: nuclease-free water or low-ionic-strength buffer (e.g., 10 mM Tris-HCl, pH 7.5–8.5) with EDTA avoided or kept minimal unless chelation of trace metals is needed outside of polymerase reactions.
• Reaction buffers: use the polymerase-supplied buffer (often containing Tris, KCl, and Mg2+). Mg2+ concentration strongly affects incorporation efficiency.
• Avoid: alcohols and most organic solvents (poor solubility), strong acids/bases (hydrolysis), and phosphate-rich buffers that may alter metal ion availability.

Miscibility and polarity (general):

• Highly hydrophilic; effectively completely miscible with water in typical working ranges; immiscible in common organic solvents.

Comparison notes:

• ddTTP (non-αS) vs ddTTPαS: both are water-soluble; αS analog can be modestly more hydrophobic, but aqueous handling is unchanged in practice.

Practical tips:

• Adjust pH to ~7.0–8.0 after dissolution using nuclease-free base/acid if needed.
• Filter (0.2 µm) into sterile, nuclease-free vials if sterility is required; do not heat-sterilize.

Storage and Reconstitution

Item-specific instructions from Product Data:

• Storage Conditions: Store at -20°C.
• Shipped In: Ice chest + Ice pads.

General guidance (informational; not a specification):

• Form: If supplied as a solid, reconstitute in nuclease-free water or 10 mM Tris-HCl (pH 7.5–8.5) to a convenient stock concentration (e.g., 10–100 mM depending on use). If supplied as a solution, verify concentration and pH on the CoA.
• Aliquoting: Dispense into single-use aliquots to avoid repeated freeze–thaw cycles. Keep on ice during handling.
• Storage: −20°C for routine storage; for long-term storage, −80°C can further limit hydrolysis. Protect from light.
• Thawing: Thaw on ice; mix gently by inversion. Avoid vortexing foamy solutions.
• Sterility: Use sterile, nuclease-free plastics and tips. If sterilization is required, filter through 0.2 µm; do not autoclave.
• Stability: Working solutions are best kept at 2–8°C for the day of use; discard if contamination or degradation is suspected.

Always consult the lot-specific CoA/Spec Sheet for exact concentration, counterion, pH, and any stabilizers present for this item.

Structure and Identity

Brief description: ddTTPαS is the α-thio analog of 2′,3′-dideoxythymidine-5′-triphosphate (ddTTP). It is a dideoxynucleotide triphosphate in which one of the non-bridging oxygens on the α-phosphate is replaced by sulfur, enabling formation of a phosphorothioate internucleotide linkage upon enzymatic incorporation.

• SKU: D1446957
• Product Name: ddTTPαS
• CAS: 158705-77-2
• Category: Nucleotide analog; chain-terminating substrate for DNA polymerases; phosphorothioate precursor
• Research Use: For research use only

Item-specific identifiers from Product Data:

• Molecular Formula: Not specified for this item; refer to CoA/Spec Sheet.
• Molecular Weight: Not specified for this item; refer to CoA/Spec Sheet.
• SMILES: Not specified for this item; refer to CoA/Spec Sheet.
• InChIKey: Not specified for this item; refer to CoA/Spec Sheet.

Structural features (general/literature):

• Nucleoside base: thymine (pyrimidine) linked to 2′,3′-dideoxyribose (both 2′ and 3′ positions lack hydroxyls; 3′-H causes chain termination).
• 5′-substituent: triphosphate chain (α, β, γ phosphates) with an α-thio modification (P=S in place of one P=O, giving diastereomeric Rp/Sp forms in typical syntheses).
• Functional groups: heteroaromatic base (amide-like), sugar (polyhydroxy motif but deoxy at 2′/3′), polyphosphate with one thio-phosphoryl.
• 2D structure in words: Thymine attached via N1–C1′ glycosidic bond to a furanose ring lacking 2′- and 3′-OH; the 5′-CH2 bears a triphosphate chain with sulfur substituting a non-bridging oxygen on the α-phosphate.

Notes:

• Exact stereochemistry at phosphorus (Rp/Sp) and counterion composition vary by supplier; consult the CoA for this specific lot.

Synthetic Utility

Role in synthesis (literature/general):

• Enzymatic DNA synthesis/extension: Incorporation by DNA polymerases enables site-specific installation of a 3′-terminal phosphorothioate linkage together with chain termination at thymine positions.
• Protecting terminal constructs: The terminal phosphorothioate resists 3′→5′ exonucleases, stabilizing DNA fragments for downstream processing or analysis.
• Mapping and footprinting: Differential cleavage of phosphorothioate linkages by chemicals or nucleases aids in mapping enzyme/DNA interactions.

Retrosynthetic considerations:

• When a DNA fragment requires a defined 3′-terminal T with phosphorothioate protection, enzymatic primer extension in the presence of ddTTPαS provides a direct route from a primed template, circumventing chemical solid-phase synthesis for that specific modification.

Integration with other transformations:

• Combine with ligases or terminal transferases in workflows where controlled termination is necessary (note: classical terminal transferase does not extend 3′-H-terminated strands).
• Pair with dNTPαS or other ddNTPαS to pattern multiple protected termini for assembly or diagnostic probe construction.

Target Specificity

Not applicable. This product is not an antibody, probe with defined biological target, or affinity reagent. It is a substrate analog for DNA polymerases used broadly in nucleic acid enzymology.