Ligation, which is a process that covalently joins the phosphate backbone of double-stranded DNA, is carried out by an enzyme called DNA ligase. DNA ligase, commonly used in biotechnology labs, was isolated from phage T4 or from E.coli. They both catalyze ligation reactions in a similar way, but they differ in cofactor requirements. The E.coli enzyme requires NAD+ while the T4 enzyme requires ATP. These cofactors are provided in buffer.

Ligation reaction

The ligation reaction generally consist of two steps:

  • DNA ends collision. This collision occurs by chance and the occurrence rate is lower in low temperature. The low temperature stabilizes the hydrogen bonding between the complementary nucleotides. The DNA ligase reaches an optimum optimum activity at 25o. A general rule is that the lower the incubation temperature, the longer the incubation time.

  • Enzymatic reaction. DNA ligase catalyzes the joining between 3'-hydroxyl to 5'-phosphate.

Ligation reaction. The AMP nucleotide is transferred to the 5'-phosphate. Then the AMP-phosphate bond is attacked by 3'-OH forming a covalent bond while releasing AMP.

The efficiency of a ligation reaction can be increased by optimizing the insert:vector ratio. A ratio of 3:1 is a good initial parameter. The concentration of DNA vector and insert can be measured using spectrophotometer such as Nanodrop. The formula required to calculate amount of gene of interest (insert) to be added in the ligation reaction is:

Example of ligation reaction optimization calculation:


Result from spectophometer measurement are:

  • DNA vector concentration: 30 ng/μL

  • Insert concentration: 15 ng/μL

We also have the data about vector and insert length which are 4000 bp and 1000 bp respectively. Optimum ratio is 3:1. We will use 60 ng of DNA vector.


(ng vector x kb size of insert)/ kb size of vector x insert/vector = ng of insert

  • (60ng x 1 kb) / 4 kb x 3/1 = 45 ng of insert
Reaction component Reaction concentration Volume
10x Ligation Buffer, T4 DNA Ligase 1X 20 μL/ 10x = 2μL
DNA vector 60ng 60ng/30ng/μL= 2μL Insert
T4 DNA Ligase 1 unit 1 μL
Total 20 μL

Plasmid vector

Expression vector contains multiple cloning sites, a selectable marker, an origin of replication, an operator and a strong promoter.

Plasmid are extra-chromosomal molecules of DNA, mostly double-stranded, covalently closed, and circular molecule. It varies in size from 1 kb to more than 200 kb. Not all plasmids are a vector. A DNA molecule such as plasmid needs to have features to be able to act as a vector for gene cloning. Generally there is two types of vectors based on their application: cloning vector and expression vector. Both vectors have the main feature that a vector should possess: a selectable marker gene, an origin of replication, and multiple cloning sites. But the expression vectors have an additional feature that is a strong promoter because it is used to express foreign protein encoded in the gene of interest. Expression vectors may contain an operator as a gene expression regulator. Operator is a segment of DNA to which a transcription factor binds. Cloning vectors are mainly used to carry and multiply the gene of interest.

A selectable marker is used to ensure that host vector retains the particular plasmid. An origin of replication gives the plasmid ability to multiply within the cell independent of the main host chromosome.

Plasmid Assembly

In principle, plasmid assembly involves these following steps:

  • Closed circular vector plasmid and gene of interest fragment are cleaved with one or more restriction enzymes.

  • Gene of interest is ligated to the linearized vector plasmid.

  • The resulting plasmid is transformed into an appropriate host.

Different restriction enzymes can generate compatible overhangs.

Higher success rate in plasmid assembly is achieved when using DNA fragment with 5' or 3' sticky/overhang ends compared with assembling plasmid with blunt end DNA fragments. DNA ligase joins two DNA fragment with compatible overhang. A DNA fragment with a particular overhang can be ligated not only to the fragment that cut with same restriction enzyme, but also to any fragment with compatible ends generated by other restriction enzymes.

Confirmation of plasmid assembly can be determined by performing DNA sequencing. It is important to confirm that the insert have been successfully ligated into plasmid vector with the correct conformation and reading frame. Frame shift can cause nonsense or misense mutation that lead to the expression of nonfunctional protein.

Restriction enzyme


Theory overview