LCR compatibility

The ligase cycling reaction, or LCR, was a protocol developed for in vitro DNA synthesis, but was adapted for in vitro DNA recombination. The ‘landmark’ paper goes into deep optimization of the procedure. Their method is rather complicated, and so there is a simpler, albeit less efficient, method as well.

 

LCR combines DNA in a modular fashion using overlapping primer(s) and phosphorylated PCR product(s). A simplified view is shown below.

Map

Primer sequences called SOCs (Scaffold Oligonucleotide Connectors) are designed to have 20bp of overlap to both DNA ends which are desired to be connected. The user then adds the DNA and SOCs together and runs a thermocycling reaction. All the DNA deanneals. Once temperatures are lowered, the primers bind both ends and bring them together. Taq ligase then ligates the 2 fragments together.

LCR-diagram_1 MapThis reaction is advantageous to gibson in that it has high efficiency for a larger number of fragments. In addition, the method is modular, with only new SOCs having to be bought for each new assembly. However, there are some drawbacks. As size increases, efficiency of the LCR reaction decreases. LCR also always requires a PCR reaction, and for simple 1 part cloning, takes 2 more primers than does SLiCE. It also can not effectively be done in a modular fashion because there are no type IIS restriction enzymes that are blunt cutters and have an over 6bp recognition sequence.

 

KG_Bricks offer compatibility with LCR with all base vectors having EcoRV sites between the Type IIS defined sites and BioBrick prefix and suffix ends. EcoRV can be used to create large amounts of vector with blunt phosphoylated ends, perfect for LCR cloning.