T4 DNA-Ligase

T4 DNA Ligase catalyzes the formation of a phosphodiester bonds between 5' phosphate and 3' hydroxyl termini in duplex DNA/RNA. 

The enzyme can join blunt end and cohesive end termini, repair single stranded nicks in duplex DNA, RNA, or DNA/RNA hybrids.

Cohesive End Ligation:

For most cohesive end ligations, a 30 minute incubation at 20°C is sufficient. Incubations at 16°C for 4-16 hours are routinely used for the majority of applications. Ligation of blunt ends and single-base pair overhang fragments requires more enzyme to achieve the same extent of ligation as cohesive end DNA fragments. Ligation may be enhanced by addition of PEG, or by reducing the rATP concentration.

- T4 DNA Ligase requires ATP > as a cofactor.
- T4 DNA Ligase is strongly inhibited by NaCL or KCl if their concentration exceeds 200mM.
- Ligation of blunt-ended and single-base pair overhang fragments requires about 50-times as much enzyme to achieve the same extent of ligations cohesive-end DNA fragments.
- Blunt-end ligation may be enhanced by addition of PEG 4000 (10% w/v) final concentration or hexamine chloride, or by reducing the ATP concentration to 50µM.
- To dilute T4 DNA Ligase for subsequent storage at -20°C the usage of a storage buffer containing 50% glycerol is recommended.
- To dilute T4 DNA Ligase for immediate use, usage of 1X Reaction Buffer is recommended.

Applications

• Cloning of restriction enzymes generated DNA fragments
• Cloning of PCR products
• Connection of double-stranded oligonucleotide linkers or adapters with DNA
• site-specific mutagenesis
• Amplified fragment length polymorphism (AFLP)
• Nicking repair in duplex DNA, RNA or DNA/RNA hybrids
• Self-circulation of linear DNA.

Definition of T4 Ligase activity

2 different definitions of T4 Ligase activities are used!

One Weiss unit is defined as the amount of enzyme required to convert 1 nmol of 32P-labeled inorganic pyrophosphate into Norit adsorbable material in 20 minutes at 37°C, using specified reaction conditions (Weiss, B., et al., (1968) J. Biol. Chem., 243, 4543). Norit is a type of activated carbon.

A Cohesive End Unit is defined as the amount of enzyme required to give 50% ligation of Hind III fragments of lambda DNA (5´ DNA termini concentration of 0.12µM (300µg/mL)) in 20µL of 1X T4 DNA Ligase Buffer in 30 minutes at 16°C.

The conversion factor between the two activities is: One Cohesive End Ligation units (CEL units) corresponds to 0.015 Weiss units. One Weiss unit therefore corresponds to 66.67 Cohesive End Ligation units (CEL units).