I am attempting to design an oligo to modify the lambda genome using ssDNA recombination roughly following the protocol of Thomason LC, Oppenheim AB, Court DL. Modifying bacteriophage lambda with recombineering. Methods Mol Biol. 2009;501:239-251. doi:10.1007/978-1-60327-164-6_21. I have read elsewhere that targeting the lagging DNA strand gives a 20fold increase in successful recombination, which is not mentioned in this protocol.
I am confused by the two methods of lambda DNA replication, first bidirectional circle-to-circle θ and then rolling-circle σ. Should I take any of this into consideration when designing my oligo in order to target the lagging strand and if so then how, or should I not overthink things and just pick a strand to design a targeting oligo for? If it helps, my mutations are in the structural genes.
Thank you for any help you can provide!
During rolling circle replication there is a single stranded stage which is more prone to homologous recombination. Recombineering relies on the the red A, B and G genes. One is an exonuclease which makes an overhang (design your oligo to complement that strand), the second is a single strand annealing protein which facilitates recombination and the third inhibits the NHEJ pathway.
Hey,
Sometimes it is really hard to know which one corresponds to the lagging and leading. You should take it into consideration though.
Order 2 oligos, one for each strand, and perform 2 recombineering experiments. When you test colonies from the two plates one will yield significantly more clones and it will be easier to find a positive one! Then you will know which one is the lagging strand for future experiments if needed.
Good luck!
Thank you Robert Adolf Brinzer for your explanation of the mechanism and Bruno Salomone Gonzalez de Castejon for your advise in how to proceed. You have been very helpful!
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