High efficiency of site-directed mutagenesis mediated by a single PCR product
High efficiency of site-directed mutagenesis mediated by a single PCR product
Xueni
Chen
1
,
Weimin
Liu
1
,
Ileana
Quinto
1,2
and
Giuseppe
Scala
1,2,
*
1
Dipartimento di Biochimica e Biotecnologie Mediche, Università `Federico II', 80131
Naples
,
Italy
and
2
Dipartimento di Medicina Sperimentale e Clinica, Università di Reggio Calabria,
Catanzaro
,
Italy
Received August 30, 1996;
Revised and Accepted November 30, 1996
ABSTRACT
We describe a highly efficient procedure for site-specific mutagenesis of double-stranded plasmids. The method relies on a single PCR primer which
incorporates both the mutations at the selection site and the desired single
base substitutions at the mutant site. This primer is annealed to the denatured
plasmid and directs the synthesis of the mutant strand. After digestion with
selection enzyme, the plasmid DNA is amplified into
Escherichia coli
strain BMH71-18 and subjected to a second digestion and amplification into the
bacterial strain DH5
[alpha]
. A mutagenesis efficiency >80% was consistently achieved in the case of two
unrelated plasmids.
Site-directed mutagenesis by unique restriction site elimination introduced by
Deng and Nickoloff allows a site-specific mutagenesis of a plasmid DNA without any subcloning step (
1
). This procedure uses two mutagenic primers: one carries the desired mutation,
the second, acting as a selection primer, carries a mutation in a unique, non-essential restriction site in the target plasmid. The mutagenesis method
relies on the simultaneous annealing of two primers (mutagenic and selection
primers) to one strand of the denatured double-stranded plasmid. After DNA elongation and ligation, the selection for
plasmids lacking the selection restriction site will also encode the desired
second site mutation. However we have found that in the case of some plasmids,
such as pGEX-BTK, the recovery of the desired mutants is much lower than that of the
selection restriction site selection mutants, yielding the desired mutant
products at a frequency of <= 10%. The reason for this low efficiency may be due to the sequence of the
target DNA, which may acquire stable secondary structures, such as stem-loops. These structures may interfere with the annealing of the mutagenic
primers and could result in low recovery of mutant plasmids. In order to
circumvent this limitation, we developed a method of PCR-fragment directed DNA synthesis. This protocol relies on the use of
selection and mutagenic primers to amplify a region of DNA lying between the
annealed selection and mutagenic primers, generating the desired mutant DNA
fragment. This mutant DNA fragment is then used as a primer for the subsequent
production of heteroduplex DNA. Since the amplified DNA fragment contains both
the desired and the selection mutations, this method results in a high
efficiency of site-directed mutagenesis.
This methodology was utilized to introduce single base substitutions into the
pGEX-BTK and pIL6-596 plasmids. pGEX-BTK contains a stop codon (TAA) corresponding to codon amino
acid (aa) 17 of the human BTK cDNA. Reversion of this codon to CAA enables the
production of a GST-BTK fusion protein. pIL6-596 plasmid carries the region of -596/+15 of the IL-6 gene, which contains a binding motif for HIV-1 Tat (
2
). The introduction of point mutations into this motif allows the identification
of single nucleotides essential for the binding of HIV-1 Tat.
