Simplifying molecular cloning
Traditional methods of molecular cloning usually involve multiple enzymatic steps, consisting of separate restriction digestion, dephosphorylation, and enzymatic ligation reactions. Alternating between the various enzymes and buffer conditions often requires purification steps, such as gel and column purification, or ethanol precipitation. These multiple steps add time and complexity to the protocol, and often result in the loss of DNA. Planning for restriction digest cloning of multiple fragments can also be time consuming, and designing larger restriction assemblies has additional challenges as larger constructs will have fewer unique restriction sites.
As with any experimental procedure, it is beneficial to reach the desired goal in as few steps as possible. This limits the potential errors due to repeated handling of samples and reagents, and avoids multiple experimental setups. The isothermal assembly method, recently developed by Gibson, et al. , greatly simplifies the process for molecular cloning of synthesized DNA molecules. Isothermal assembly also makes it possible to include larger, more complex assemblies than traditional cloning methods.
How isothermal assembly works
In a single reaction, isothermal assembly combines several overlapping DNA fragments to produce a ligated plasmid ready for transformation. The method relies on use of an enzyme mixture consisting of the mesophilic 5’ T5 exonuclease, a thermophilic DNA ligase, and a thermophilic proofreading DNA polymerase. Details of what happens in the reaction are outlined in Figure 1.
Figure 1. Isothermal assembly method for double-stranded DNA. The following events take place in a single 50°C reaction. 1) Individual segments of dsDNA are designed so that the 3’ strands have complementary overlaps. 2) A mesophilic 5’ exonuclease briefly digests into the 5’ ends of the doublestranded DNA fragments before being inactivated at 50°C. 3) The newly generated complementary 3’ overhangs anneal. 4) A high fidelity DNA polymerase fills in gaps completing fragment-containing circular plasmids and leaving free ends retracted on linear fragments. 5) Finally, a thermophilic DNA ligase covalently joins DNA segments.
When added to DNA and placed at high temperatures, the exonuclease digests the double-stranded DNA from its 5’ ends. This reaction is quickly inactivated at 50°C, resulting in 5’ single-stranded ends without completely degrading either DNA strand. (Because it is a 5’ exonuclease, the T5 activity does not compete with DNA polymerase and the newly created overhangs are not immediately filled in when the exonuclease is inactivated.)
For optimum assembly and ligation, the ends of the DNA sequences are designed to overlap by at least 30 bases. The brief 5’ exonuclease activity exposes the overlapping bases, leaving variable length overhangs at the 3’ ends. These large overlaps are beneficial for joining fragments as they allow for highly specific hybridization of DNA fragments—compared to a typical restriction cloning reaction in which complementary overhangs are no more than 4 bases.
Because the T5 exonuclease processes a random number of bases before being inactivated there will be single-stranded gaps between the hybridized 3’ ends and the remaining 5’ ends. The high-fidelity DNA polymerase fills the resulting gaps and the thermophilic DNA ligase fuses the fragments together.
A major benefit of the isothermal assembly method is that it has fewer specific design requirements than other forms of cloning. For example there is no need to look for endogenous recognition sites for restriction enzymes, or to insert such sites that could subsequently affect the usefulness of the final DNA construct. Therefore, the basic principles of design for isothermal are fairly simple:
- For the isothermal assembly to work the ends of the vector and the insert(s) must complement one another. In general 30 bases of complementarity are optimal at each end. Ideally the ends should be free from secondary structure.
- If you are planning to assemble DNA that has been produced from PCR, primers can be designed so that each primer has 15 bases of overlap into what will be the adjacent fragment to create the 30 bases of overlap.
- Since hybridization of long 3’ overhangs is similar to hybridization of PCR primers, avoid designing 3’ overlaps in regions with repeated DNA motifs or repeated bases that can hybridize though improperly aligned.
- If the final construct is to be linear rather than a fragment subcloned into a plasmid vector, the ends of the DNA will be retracted at each 5’ end. If desired, these ends can be filled in a separate reaction using a short complementary oligonucleotide primer, complementary to the 3’end. This is not necessary for circular constructs, as all gaps are filled and the ends ligated during the isothermal reaction.