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Cat. No.: PBCV-1250 (for 1250U)

Cat. No.: PBCV-5k (for 5KU)

The lyophilized PBCV-1 DNA Ligase (SplintR Ligase) is also available by inquiry, which can be transported at room temperature.

We also offer DNA-Free PBCV-1 DNA Ligase, which has undergone a rigorous E. coli DNA removal process.

Description

PBCV-1 DNA Ligase, also known as PBCV DNA Ligase, SplintR™ Ligase, or Chlorella virus DNA Ligase, is an ATP-dependent DNA ligase that efficiently catalyzes the ligation reaction between two adjacent DNA single strands that are complementary to a single-stranded RNA. In this process, one of the adjacent DNA strands that provides a 3' hydroxyl group is called the acceptor DNA strand, while the other strand that provides a 5' phosphate group is called the donor DNA strand. During this ligation process, an complementary RNA strand acts as a "splint" or "scaffold" to hold the two DNA single strands together.

PBCV-1 DNA Ligase can tolerate various combinations of base pairs 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. Further inhibition of enzyme activity occurs when both the first and second base pairs formed between the donor DNA and the "splint" RNA are dC/G or dG/C base pairs. The ligation efficiency is higher 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 has a higher affinity (Km = ~1nM) for DNA substrates immobilized by RNA "splints" compared to T4 DNA ligase. When combined with subsequent techniques such as PCR, qPCR, or rolling circle amplification, it enables the ultra-sensitive detection of specific target RNAs at sub-nanomolar levels in complex mixtures. The activity of PBCV-1 DNA Ligase allows for qualitative or quantitative detection of various RNAs, including miRNAs, mRNAs, and non-coding RNAs, including SNPs. It is also used in next-generation sequencing and molecular diagnostics.

PBCV-1 DNA Ligase has a broad range of ATP concentration requirements, with effective activity observed at ATP concentrations ranging from 10µM to 1mM. It also exhibits high tolerance to pH, performing well within the pH range of 6.5 to 9. Typically, the enzyme shows higher activity when the concentration of Mg2+ is greater than 5mM and the pH is between 7.5 and 8.0. The activity of the enzyme can be enhanced by increasing the reaction temperature to 37℃ and supplementing with 5mM Mn2+. However, the enzyme activity is inhibited when the salt ion concentration in the reaction system exceeds 100mM.

Application

PBCV-1 DNA Ligase is used for the detection of microRNA and other small RNAs using probe-based methods; for the connection of single-stranded DNA through complementary RNA sequences using a "splint" or "chimeric" approach; for the connection of specific RNAs using DNA probes; for SNP or alternative splicing detection; and for RASL-seq analysis.

Source

The PBCV-1 DNA Ligase gene is expressed and purified from E. coli strains.

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 20μl reaction at 25°C in 15 minutes using 1X Ligation Buffer.

Purity

The enzyme does not contain DNA endonucleases, exonucleases, RNAases, or phosphatases.

Enzyme storage buffer

10mM Tris-HCl (pH 7.4, 25°C), 300mM NaCl, 1mM DTT, 0.1mM EDTA, 50% (v/v) glycerol.

10X Reaction Buffer

500mM Tris-HCl (pH 7.5, 25°C), 100mM MgCl2, 10mM ATP, 100mM DTT.

Inactivation or inhibition

Heating at 65°C for 20 minutes.

Precautions

• PBCV-1 DNA Ligase is inhibited by monovalent cations. It is recommended to keep common salts such as NaCl and KCl below 50mM in the reaction. To maintain the stability of the enzyme, it is provided in a storage buffer containing 300mM NaCl. When adding the enzyme to the reaction system, at least a 6-fold dilution is recommended, with dilutions greater than 10-fold being preferable.
• The recommended initial experimental temperature for PBCV-1 DNA Ligase is between 16-37°C, with 25°C being a suitable reaction temperature. The ligation reaction time for the enzyme typically ranges from 10-60 minutes, but for many experiments, a recommended reaction time of 15 minutes is sufficient.
• The final concentration of PBCV-1 DNA Ligase in the reaction system is suggested to be between 100nM-1µM. The concentration of PBCV-1 DNA Ligase produced by Biyun Tian is approximately 13µM. It is advised to set the enzyme concentration at least 2-3 times higher than the substrate concentration in the ligation reaction.
• If the reaction is not proceeding as expected, it is recommended to extend the incubation time rather than increasing the enzyme concentration above 1µM.
• For substrates with low ligation efficiency in the standard PBCV-1 DNA Ligase reaction buffer, such as those with G:C base pairs at the ligation site, adjusting the final concentration of ATP to 10µM is recommended to improve ligation efficiency.
• Increasing the reaction temperature can enhance the activity and specificity of PBCV-1 DNA Ligase. A reaction temperature of 37℃ can improve the detection efficiency of microRNA and other small RNAs.
• The ligation efficiency of PBCV-1 DNA Ligase gradually decreases with decreasing length of the "splint" RNA (from 50bp to 20bp). When the length of the RNA "splint" is equal to or less than 10nt, the ligation efficiency is essentially zero.
• This product is intended for scientific research by professionals only and should not be used for clinical diagnosis or treatment, or for food or drug purposes. It should not be stored in residential premises.
• For your safety and health, please wear laboratory attire and disposable gloves when handling.

Storage

Store at -20°C.

DNA Ligase Products:

• T4 DNA Ligase
• Rapid T4 DNA Ligase
• T4 DNA Ligase (Fast)
• T7 DNA Ligase
• E. coli DNA Ligase
• PBCV-1 DNA Ligase
• T3 DNA Ligase
• Taq DNA Ligase
• Pfu DNA Ligase
• High-Fidelity DNA Ligase
• High-Salt DNA Ligase

SBS Genetech is recognized as one of the global major leading industry players in Ligase Market by third-party market researchers. For more details, please visit Ligase Market - A Global and Regional Analysis Focus on Product, Source, Application, End User, and Country - Analysis and Forecast, 2022-2032