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Cat. No.: 5DRA-3k (for 3KU)

Cat. No.: 5DRA-15k (for 15KU)

Description

5' DNA/RNA Adenylase, also known as Single-Strand Nucleic Acid 5' Adenylase or single-strand nucleic acid 5' adenylation enzyme, catalyzes the conversion of 5'-phosphorylated single-stranded DNA or single-stranded RNA (pDNA or pRNA) to generate 5'-adenylated DNA (AppDNA) or 5'-adenylated RNA (AppRNA). Regardless of whether the 3' end of the DNA or RNA substrate is modified with amino groups or other blocking agents, this product efficiently catalyzes the production of 5'-adenylated products. Under conditions where ATP is absent in the reaction system, this product can also catalyze the formation of circular DNA from 5'-phosphorylated single-stranded DNA (pDNA).

For the 5' adenylation modification reaction using 5' DNA/RNA Adenylase, ATP and 5'-phosphorylated single-stranded DNA or single-stranded RNA to be adenylation-modified are required.

The principle of 5' DNA/RNA Adenylase catalyzing the adenylation reaction of single-stranded nucleic acids at the 5' end is as follows: Starting with 5'-phosphorylated single-stranded DNA or single-stranded RNA as the substrate, this product first hydrolyzes ATP into AMP and PPi, then transfers AMP to the 5' phosphate group of the single-stranded DNA or RNA, resulting in the formation of 5'-adenylated single-stranded DNA or RNA and generating adenylation products.

Application

This enzyme is used to prepare 5'-adenylated single-stranded DNA adapters, which are ultimately used to add adapters to the 3' end of RNA (such as miRNA) with a hydroxyl group at the 3' end, or single-stranded DNA with a hydroxyl group at the 3' end, for applications such as cloning, high-throughput sequencing library construction, or PCR detection.

Source

The adenylase enzyme is derived from a thermophilic bacterium and is expressed and purified in Escherichia coli.

Unit Definition

One unit is the amount of enzyme required to catalyze the transfer of 1 pmol of AMP to a pDNA substrate in a total reaction volume of 20 μl within 15 minutes at 65°C.

Purity

It does not contain DNA endonucleases or exonucleases, and it is free of ribonucleases.

Inactivation or Inhibition

Heating the 5' DNA/RNA Adenylase at 85°C for 5 minutes will result in its inactivation.

When the reaction volume is 20 μl, the 3kU and 15kU packaging sizes of this product can be used for 40 and 200 reactions of 5'-end DNA/RNA adenylation, respectively.

Precautions

• Phosphorylation of the 5' end of the substrate single-stranded DNA or RNA is necessary, while the 3' end can be amino-modified or left unmodified. When not performing 3' end amino modification, it is recommended to increase the ATP concentration to 0.5 mM to prevent substrate circularization or concatenation reactions.
• 5' DNA/RNA adenylase cannot ligate nicks between double-stranded DNA (dsDNA).
• The optimal reaction temperature for 5' DNA/RNA adenylase is 65℃. At 25℃, there will be de-adenylation phenomenon. Therefore, it is recommended to incubate the reaction at 85℃ for 5 minutes to inactivate the 5' DNA/RNA adenylase after completion of the reaction. If the 5' DNA/RNA adenylase is not adequately inactivated, subsequent exposure to room temperature conditions may lead to a decrease in adenylation efficiency.
• Store the 5' DNA/RNA adenylase in an icebox or on an ice bath during use, and immediately store it at -20℃ after use.
• This product is intended for scientific research by professionals only and must not be used for clinical diagnosis or treatment, food or drug purposes, or stored in residential premises.
• For your safety and health, please wear laboratory attire and disposable gloves when handling.

Storage

The minimum shelf life is 2 years at -20°C.