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KLD Site-Directed Mutagenesis Protocol

Site-Directed Mutagenesis using WVA, back-to-back primers and KLD
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KLD Site-Directed Mutagenesis Protocol using Back-to-Back Primers

Advantages of KLD Site-Directed Mutagenesis

  • Simple to use KLD mix
  • High-yield exponential PCR.
  • Simple primer design.
  • In vitro digestion of the template DNA.
  • 90% of clones contain the desired mutation.
  • Suitable for insertions of ∼ 80 bases or longer if using oligos > 60-mers.
  • Ideal for creating large deletions.
  • Easy 3-step workflow***.

Disadvantages

  • Long PCR protocol – 25 cycles (between 4 and 8 hours or 1 to 2 hours using Fast & Steep PCR).
  • Only 1 primer contains the mutation which may generate non-methylated and non-mutated PCR products.
  • Primers or Dpn I-generated fragments are likely to be inserted at the ligation site.

***To ensure that no extra mutations are introduced in the vector backbone, site-directed mutagenesis strategies, such as the herein KLD strategy, which use whole vector amplification require subcloning of the sequenced insert into a vector that hasn’t been amplified by PCR. Alternatively, the whole vector can be sequenced.

Back-to-back primer site-directed mutagenesis with KLD

STEP 1 - Primer Design

Point Mutation

As in the name of the technique and in contrast to QuickChange and Fast Mutagenesis primer design, both primers must be designed ‘back-to-back’ in opposite directions. Only one (1) of the primers must contain the desired point mutation.

Back-to-back SDM with KLD - point mutation primer design

Deletion

The back-to-back primer design allows for deletions of unlimited size to be generated simply by positioning both 5′ ends of forward and reverse primers directly on the sequence flanking the desired deletion.

Back-to-back SDM with KLD - deletion primer design

Insertion

Both primers must be designed ‘back-to-back’ in opposite directions and either one of them must contain the sequence to be inserted on its 5′ end. The insertion can be split between both forward and reverse primers if desired.