L8 DNA&RNA Ligase

一、The function of an DNA ligase

Definition:

• DNA ligases catalyze the formation of a phosphodiester bond between
• DNA single strands in the duplex form.

What’s the function of DNA ligase in vivo ?

1. DNA damage repair
2. DNA recombination during Meiosis and antibody formation
3. Seal the gaps during DNA replication

三、DNA ligase & RNA ligase

T4 DNA ligase

Origin: T4 Phage

Feature:

1. Favor dsDNA over ssDNA. Much less efficient in ssDNA ligation
2. Use ATP as energy source

**Usage: **

1. Repair the single stranded nick in the double stranded DNA or RNA
2. Join blunt end and cohesive end termini

E.coli DNA ligase

Origin: E.coli

Feature:

1. Use dsDNA as substrate, much less efficient on ssDNA
2. Use NAD as a cofactor and energy source
3. Only repair cohensive end but not blunt end

Usage:

• Selective ligation of nicks in dsDNA

T4 RNA ligase I

Origin: T4 phage

Feature:

1. It is a single stranded ligase
2. Substrate include both ssDNA and ssRNA

Usage:

• Ligate a ssDNA/ssRNA with a ssDNA or ssRNA adaptor (NGS library prep）

T4 RNA ligase 2

Origin: T4 phage

Feature:

• It is a double-stranded RNA ligase

Usage:

1. Seal nicks in dsRNA.
2. Ligate the 3’OH of RNA to the 5’ phosphate of DNA in a double stranded structure

	dsDNA	ssDNA	dsRNA	ssRNA	Comments
T4 DNA ligase	✔	✔, but much less efficient	X	X	1. ATP as energy source 2. Joint blunt and cohesive end
E.coli DNA ligase	✔	✔, but much less efficient	X	X	1. NAD as energy source 2. Joint cohesive end
T4 RNA ligase 1	X	✔	X	✔
T4 RNA ligase 2	X	X	✔	X

四、Pre-adenylated adaptor

5’ Pre-adenylated adaptor

DNA/RNA-adenylate is a intermediate of ligation reaction

Pre-adenylated 5’ end can increase ligation efficiency of T4 DNA/RNA ligase significantly

rAPP-Adaptor:

五、Consideration for a ligation reaction

1. Choose the right ligase according to the substrate
2. Ligation temperature, time and buffer composition
3. Check if 3’OH and 5’ P exist in your substrate

Some primers do not have 3’-OH or 5’-P, therefore the PCR product do not has these functional groups in the 5’ and 3’ end too. Therefore, there will exist a nick after ligation reaction (hard for ligation).