The Isothermal Assembly Reaction: My Cloning Hero

I was trying to make a plasmid that had various parts from five other plasmids. Normally, I’d say “good luck” to anyone trying a three point ligation and so I considered a five point ligation to be a bit beyond mere mortals such as myself. But with the isothermal assembly reaction, the lab downstairs has had good luck with three and four point ligations. So it was worth a shot…

The isothermal assembly technique uses three enzymes: a 3′ exonuclease, taq ligase, and pfu polymerase. Basically, you do PCR using primers such that each successive piece that you want to stitch together has about 20-30 bp of homology to the next piece. i.e the PCR primers should be homologous to both the region you are amplifying and also the next piece on either end.

After you gel purify the PCR fragments (restriction digested samples also work as long as there is sufficient homology on either end), you mix them together and add them to the enzyme mixture and then incubate at 50°C for one hour. The reaction is such that the 3′ exonuclease will start degrading each of the fragments for about 5-10 minutes until the high temperature diminishes its activity. The fragments will now anneal by base pairing between the homologous regions with gaps. Then the thermostable enzymes, pfu polymerase and taq ligase, will fill in and ligate the construct. Then you transform competent bacteria as usual and check colonies for properly reassembled plasmids.

I was expecting about 1:100 efficiency for a five point ligation, but I rolled the dice and started screening with just 6 colonies to see if I could get lucky and get one good one from that. To my surprise, three of the six colonies showed the correct size and restriction pattern using two sets of diagnostic restriction cuts. I sequenced two of them using the same primers that I used for PCR and that provided about 90% sequence coverage. The sequences showed that the constructs were correct.

I was amazed that ~50% of the 100-200 colonies after this five point ligation were correct. I will now make up the enzyme mix for the rest of the lab to use so we don’t have to keep stealing it from the lab downstairs…

I would definitely recommend isothermal assembly to anyone attempting a difficult cloning project.

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The isothermal assembly reaction protocol from the Megason lab wiki page:

1. PCR up your fragments of choice, and gel purify.
2. Not exceeding a total volume of 5 ul, in a PCR tube, combine fragments at equal molecular ratio [e.g. amount fragment1 = 100 ng * (fragment size 1/ fragment size 2); amount fragment2 = 100 ng * (fragment size 2/ fragment size 1) … etc.]. If required, bring to 5 ul with ddH2O. I recommend using approx. 100 ng of plasmid backbone (fragment containing antibiotic resistance).
3. Add the combined fragments (5 ul) to 1 Isothermal Assembly reaction aliquot (15 ul) and mix by pipetting (20 ul total).
4. Place rxn. at 50C for 15 -60 min.
5. (optional for chem. transform.) Purify with Qiagen PCR purification (MinElute) kit. Elute in 20 ul of ddH2O.
6. Transform with 1 ul of assembly rxn.

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Reference: Nature Methods (Vol.6 No.5, May 2009, pp343-345)

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Michael Gage obtained his Ph.D. in Pharmaceutical Chemistry from the University of California at San Francisco where he studied the role of cytoplasmic protein motifs in the membrane recycling of G-protein coupled receptors. He currently works in the Kirschner Lab at Harvard University exploring the use of mass spectrometry to solve biological problems.
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Looking to screen colonies faster? Learn to perform colony PCR with this video and accompanying text protocol.

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Have any isothermal assembly or cloning heroism you’d like to share?

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