PBCV-1 DNA Ligase produced by Aladdin is an ATP-dependent DNA ligase that efficiently catalyzes the ligation of two adjacent DNA strands annealed to a complementary single-stranded RNA. Of these two adjacent DNA strands, the one providing a 3'-hydroxyl group is referred to as the acceptor DNA, and the one providing a 5'-phosphate group is referred to as the donor DNA. This ligation reaction requires a complementary RNA strand to act as a “splint” or “scaffold” for the two DNA strands. PBCV-1 DNA Ligase can tolerate a variety of base-pair combinations at the ligation junction. However, partial inhibition occurs when the first base pair formed between the 5'-phosphorylated donor DNA and the splint RNA is dC/G or dG/C. Enzyme activity is further inhibited when both the 1st and 2nd base pairs between the donor DNA and the splint RNA are dC/G or dG/C. Higher ligation efficiency is achieved when the first base of the 5'-phosphorylated donor DNA is dA or dT, and when the first base of the 3'-hydroxylated acceptor DNA is dT. This enzyme exhibits stronger affinity than T4 DNA Ligase for RNA splint-mediated DNA ligation (Km ≈ 1 nM). Combined with downstream techniques such as PCR, qPCR, or rolling circle amplification, it enables ultra-sensitive detection of specific target RNAs at sub-nanomolar levels in complex mixtures. This activity of PBCV-1 DNA Ligase allows its use in the qualitative or quantitative detection of miRNA, mRNA, and non-coding RNAs including various SNPs, as well as in next-generation sequencing and molecular diagnostics. PBCV-1 DNA Ligase has a broad requirement for ATP concentration and functions effectively from 10 μM to 1 mM. It also has high pH tolerance and performs well within the range of pH 6.5–8.9. Generally, PBCV-1 DNA Ligase shows high activity when the concentration of Mg²⁺ is above 5 mM and the pH is between 7.5 and 8.0. Its activity can be enhanced by increasing the reaction temperature to 37 ℃ and supplementing with 5 mM Mn²⁺. However, the enzyme activity is inhibited when the salt ion concentration in the reaction system exceeds 100 mM.
| Source | Recombinant expressed in Escherichia coli
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| Appearance | Sterile liquid
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| Storage Buffer | 10mM Tris-HCl, 300mM NaCl, 1 mM DTT, 0.1 mM EDTA, 50% (v/v) Glycerol, pH 7.4, 25℃.
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| Enzyme Concentration | 25 U/μl |
| Purity | Free of DNA endonucleases, exonucleases, RNases and phosphatases.
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| Activity Definition | One unit is defined as the amount of enzyme needed to ligate (to 50% completion) 2 picomoles of a tripartite FAM-labeled DNA:RNA hybrid substrate in a 20 μl reaction at 25°C in 15 minutes in 1× Ligation Buffer.
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Component List
P748845
| Component | 1250U
| 5KU
| Storage |
| P748845A | PBCV-1 DNA Ligase (25U/μl)
| 50µl | 200µl | -20℃. Avoid freeze/ Thaw cycle. |
| P748845B | 10× Reaction Buffer | 150µl
| 600µl | -20℃. Avoid freeze/ Thaw cycle. |
Product Applications
Probe-based detection of small RNAs such as microRNAs: ligation of single-stranded DNA "splinted" by complementary RNA sequences; detection of specific RNAs via ligation using DNA probes; detection of SNPs or alternative splicing; RASL-seq analysis.
Product Advantages
PBCV-1 DNA Ligase can tolerate a variety of base-pair combinations at the ligation junction, but partial inhibition occurs when the first base pair formed between the 5'-phosphorylated donor DNA and the splint RNA is dC/G or dG/C.
Instructions for Use
1. Preparation of the hybrid DNA/RNA duplex substrate:
Mix two single-stranded DNA molecules and one complementary single-stranded RNA molecule in equimolar amounts. The recommended final concentration is 20 μM, with a working range of 10–50 μM. Incubate at 90°C for 1 minute, then gradually cool to 25°C to anneal and form the DNA/RNA hybrid duplex. It is recommended to use the supplied Annealing Buffer for RNA oligos (5×) and follow the product instructions for the annealing reaction. If the annealed duplex is not used immediately, it is recommended to store it at -80°C.
2. For the ligation of a nick between two DNA strands in a DNA/RNA hybrid, prepare the reaction mixture on ice according to the following table:
| Reagent | Volume | Final Concentration |
|---|
| DEPC-treated Water | 15 μl | - |
| 10× Reaction buffer | 2 μl | 1× |
| Nicked DNA/RNA Substrate (1 μM) | 2 μl | 0.1 μM |
| PBCV-1 DNA Ligase (25 U/μl) | 1 μl | 1.25 U/μl |
| Total Volume | 20 μl | - |
Note: Since RNA handling is involved, strict RNA protocols must be followed to avoid RNase contamination. All related reagents and consumables must be RNase-free or treated with DEPC to eliminate RNase.
3. Ligation: Incubate at 25°C for 15–60 min.
4. Termination: Incubate at 65°C for 20 min, or inactivate by adding EDTA.
5. Storage: Place the reacted sample on ice for short-term use, or store directly at -20°C.
Precautions
1. PBCV-1 DNA Ligase is inhibited by monovalent cations. Common salts such as NaCl and KCl should be kept below 50 mM in the reaction. For storage stability, the enzyme is supplied in a storage buffer containing 300 mM NaCl. When added to the reaction system, the enzyme should be diluted at least 6-fold, preferably more than 10-fold.
2. The recommended initial reaction temperature for PBCV-1 DNA Ligase is 16–37°C, with 25°C as the suggested setting. The ligation time is typically 10–60 min, and 15 min is recommended for most applications.
3. The recommended final concentration of PBCV-1 DNA Ligase in the reaction is 100 nM–1 μM. The supplied PBCV-1 DNA Ligase is approximately 13 μM. It is advised to use the enzyme at 2–3 times the substrate concentration.
4. If the reaction does not proceed efficiently as expected, extend the incubation time rather than increasing the enzyme concentration above 1 μM.
5. For substrates with low ligation efficiency in the standard PBCV-1 DNA Ligase reaction buffer (e.g., those with G:C base pairs at the ligation junction), adjusting the ATP concentration to 10 μM is recommended to improve ligation efficiency.
6. Increasing the reaction temperature enhances the activity and ligation specificity of PBCV-1 DNA Ligase. A reaction temperature of 37°C improves the detection of small RNAs such as microRNAs.
7. Ligation efficiency of PBCV-1 DNA Ligase gradually decreases as the splint RNA length is reduced (50 bp to 20 bp). Ligation activity is nearly abolished when the splint RNA length is ≤ 10 nt.
8. This product is for scientific research use by qualified personnel only. It is not intended for clinical diagnosis or treatment, food or pharmaceutical applications, and must not be stored in ordinary residential premises.
9. For your safety and health, please wear a lab coat and disposable gloves during operation.
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