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DNA molecules are connected to each other
T4 phage DNA ligase differs from E. coli DNA ligase in its ability to efficiently ligate blunt-ended DNA fragments (Sgaramella and Khorana, 1972; Sgaramella and Ehrlich, 1978). This feature makes it particularly valuable, as blunt ends are commonly generated during DNA manipulation. Unlike sticky-end ligation, which relies on complementary base pairing, T4 ligase can connect any two DNA molecules that have been made blunt, offering a more flexible approach in molecular cloning.
One of the key advantages of using T4 DNA ligase for blunt-end ligation is that it eliminates the issue of mismatched sticky ends. This means that theoretically, any two DNA sequences can be joined together, provided the proper enzymatic conditions are met. Additionally, when blunt ends are connected, they may create new restriction enzyme recognition sites, which can be useful for further cloning steps or screening.
However, there are some disadvantages to consider. First, the efficiency of blunt-end ligation is generally lower than that of sticky-end ligation. One reason for this is the absence of complementary base pairing to stabilize the connection, and another is that T4 DNA ligase has a much higher Km value for blunt ends compared to sticky ends—often around 1000 times higher. Second, because the ligation process is not directional, the inserted fragment can be oriented in either direction, which may complicate downstream analysis. To address this, researchers often include unique restriction sites in both the vector and the insert to help identify the correct orientation.
Another potential issue is the possibility of multiple insertions. When working with blunt ends, it's possible for several copies of the target fragment to be inserted into the vector, and each copy might be oriented differently. This can lead to complex results that are harder to interpret. Fortunately, larger DNA fragments tend to reduce the likelihood of multiple insertions due to their lower concentration in the reaction mixture.
To achieve successful blunt-end ligation, certain conditions must be met. The reaction requires a low concentration of ATP (around 0.5 mmol/L), as higher concentrations can inhibit the enzyme. It’s also important to avoid polyamines like spermidine, which can interfere with the ligation process. A high concentration of T4 DNA ligase—typically around 50 Weiss units per milliliter—is necessary to compensate for the lower efficiency of blunt-end joining. Finally, the DNA fragments should be present at a relatively high concentration to increase the chances of successful ligation.