'Round-the-horn site-directed mutagenesis

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‘Round the Horn Site-directed mutagenesis

Submitted by Smoore



 I was in grad school and had to make several codon substitutions at the same position in a cloned gene.  I came up with this protocol and called it "Around-the-Horn" a phrase used in baseball meaning to throw that ball through all of the base positions.  The phrase originally referred to "Rounding Cape Horn of South America" a very difficult, but rewarding journey ("Round-the-horn" is not difficult).  Subsequently, a group published a paper describing the process, but I was first and take full credit and have used it in ways not described by others.
  • The Size of the muation is limited to a few base pairs.


Primer design




The reverse primer in theis example sits on the complementary strand and abuts the site of mutation.



  • Primers without mutations at their 5' ends can be spaced apart on the template with a gap between them to make deletions of a desired size.
  • You can amplify a low-copy plasmid with primers that introduce cloning sites for receiving an insert. The PCR product becomes the "vector" component of the ligation reaction. In this case, you don't need to phosphorylate the primers because the restriction digest of the ends will leave 5' phosphates. An advantage using PCR to prepare your vector is that background "vector re-ligation" reactions are practically eliminated. Remember to transform bacteria that have no restriction system as the majority of the plasmid will me unmodified.

Primer Phosphorylation

Primer stock at 100 μM in water or TE. For each primer:

37 μL water 5 μL 10 Kinase reaction buffer (comes with PNK) 1 μL 50 mM MgSO4 (comes with most polymerases) 5 μL primer (10 μM final) 1 μL 100 mM ATP 1 μL PNK 50

37 degrees for 30-60 minutes.

Heat each reaction to kill the PNK (65 degrees for 20 minutes, or 95 degrees for 5 min).


The Mixture

Set up on ice. After mixing, put into pre-heated PCR block.

39 μL water 5 μL 10X polymerase buffer 1.5 μL forward primer (0.3 μM final) 1.5 μL reverse primer 1 μL dNTPs (20 mM each) 1 μL template (mini-prep’d plasmid) 1 μL polymerase (not Taq, use Vent, Deep Vent, 9°N, Pfu) 50 μL

PCR program

(1) 95º 1’ (2) 93º 30” (3) 55º 30” (~5º below Tm) (4) 72º 2’ per kb being amplified (5) Goto (2) 25 more times (6) 4º hold

Product Preparation



Product ligation

2.3 μL water* 2 μL PCR product 0.5 μL 10X ligation buffer 0.2 μL ligase 5 μL

  • If there is not much product, replace the water fraction with more PCR product.

Incubate over night at room temp. These Blunt ligations can be pretty slow. Some enzymes (e.g. 9° North and Taq) leave crap on the 3' ends. This greatly reduces the efficiency of the blunt-end ligations.



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