Ligation-During-Amplification

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Ligation-During-Amplification ( LDA ) is a molecular biological method for the linear amplification of circular DNA ( e.g. a plasmid ) in a PCR- based process. One or more sequence-specific primer pairs with the sequence changes to be introduced, a thermostable DNA polymerase and a thermostable DNA ligase are used. This method is also marketed as QuikChange Multi by Stratagene.

This method is used for the targeted mutagenesis of plasmids in the course of a desired adaptation of the vector or the expression cassette in the course of the protein design . A mutagenic primer is used to linearly amplify and ligate a strand of circular plasmid DNA (template) in a thermal cycler in several cycles . Then, the parental, methylated plasmid DNA (template) with which it is restriction enzyme Dpn I, which only dam methylated DNA cleave degraded. The amplified, mutated, single-stranded, circular DNA is then transformed into bacteria and completed there to form double-stranded plasmid DNA.

Another method is based on the reverse of the principle. To this end, hydroxymethyl-deoxycytidine (HMdCTP) is added instead of the dCTP for amplification and then restricted with a mixture of the restriction endonucleases ApeK1 and Pho1 in an activity ratio of 2: 1. As a result, only the DNA template (without hydroxymethyl deoxycytidine) is broken down. The ligation takes place after the transformation in vivo .

literature

  1. ^ Z. Chen, DE Ruffner: Amplification of closed circular DNA in vitro . In: Nucleic Acids Res , Volume 26, 1998, pp. 1126-1127, PMID 9461478 , PMC 147341 (free full text)
  2. ^ AR Shenoy, SS Visweswariah: Site-directed mutagenesis using a single mutagenic oligonucleotide and DpnI digestion of template DNA. In: Anal. Biochem. Volume 319, 2003, pp. 335-336. PMID 12871732 doi: 10.1016 / S0003-2697 (03) 00286-0
  3. ↑ A. Sawano, A. Miyawaki: Directed evolution of green fluorescent protein by a new versatile PCR strategy for site-directed and semi-random mutagenesis . In: Nucleic Acids Res , Vol. 28, 2000, p. E78. PMID 10931937 , PMC 108465 (free full text)
  4. ↑ QuikChange Multi Site-Directed Mutagenesis Kit. Manual ( Memento from May 23, 2006 in the Internet Archive ; PDF)

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