Biotechnology Letters 22: 1603–1609, 2000.
© 2000 Kluwer Academic Publishers. Printed in the Netherlands.
1603
Optimisation of overlap extension PCR-based mutagenesis of a GC-rich
DNA template: application to the human α
2C
-adrenoceptor cDNA
T. Wurch, F. Lestienne & P.J. Pauwels
∗
Department of Cellular and Molecular Biology, Centre de Recherche Pierre Fabre, 17, Av. Jean Moulin,
81106 Castres C´edex, France
∗
Author for correspondence (Fax: +33 563714363; E-mail: peter.pauwels@pierre-fabre.com)
Received 26 June 2000; Revisions requested 12 July 2000; Revisions received 11 August 2000; Accepted 11 August 2000
Key words: α
2C
-adrenoceptor, GC-rich DNA sequence, overlap extension PCR
Abstract
PCR-based overlap extension mutagenesis was applied to introduce a Thr
381
to Lys mutation in the α
2C
-
adrenoceptor (α
2C
AR) coding sequence. This cDNA contains 71% G+C nucleotides and conventional PCR
procedures were inefficient in generating a corresponding amplification product. Only the combined use of a pre-
PCR denaturation step at 100
◦
C followedby quick chilling on ice and the addition of 1 M of a commercial GC Melt
reagent and 5% (v/v) dimethylsulphoxide with the Advantage GC cDNA PCR kit yielded efficient amplification
during the three successive PCR steps of the overlap extension procedure. Transient expression of the mutant
Thr
381
Lys α
2C
AR in Cos-7 cells was performed to determine the binding profile for a series of α
2
AR ligands
using [
3
H]RX 821002.
Introduction
Polymerase Chain Reaction (PCR) is a widely ap-
pliedmolecular biology technique:it allows numerous
and different applications going from cloning, DNA
sequencing, mutagenesis to detection of single nu-
cleotide polymorphisms (Gelfand & White 1990). Al-
though theoretically all types of template DNA can
be amplified by PCR, DNA sequences which are
composed of a high guanosine/cytosine nucleotides
(GC) content, very often require laborious and time-
consuming work in the optimisation of the amplifi-
cation conditions. The nuclear genomes of mammals
contain approximately41% G+C nucleotides, and the
CGand GC dinucleotidesare about five-foldmore rare
than expected from the G+C content (Normore et al.
1976). Nevertheless, GC sequences are unevenly dis-
tributed in the genome. They may be present at 10-
to 20-times above their average density in selected
regions defined as GC islands, for which the G+C
content represents more than 50% and that are 1000
to 2000 nucleotides long (Antequera & Bird 1993a).
Many mammalian genes are associated with such GC
islands (Antequera & Bird 1993b). Quantification of
the number of GC islands per haploid genome in hu-
mans has been estimated to about 45000 for a total
number of 80000 genes (Antequera et al. 1990).
We were interested to introduce a mutation at
the Thr
381
position of the human α
2C
-adrenoceptor
(α
2C
AR, RC: 2.1.ADR.A2C) coding sequence using
a modified overlap extension PCR technique (Wurch
et al. 1998). The reported wild-type (wt) nucleotide
sequence (Regan et al. 1988) indicated a G+C nu-
cleotide content of 71% overthe entire α
2C
AR coding
region. We hereby describe different PCR conditions
and additivesto the amplification reaction, which were
applied to amplify this nucleotide sequence and intro-
duce the respective mutation. Additives like dimethyl-
sulphoxide (DMSO), glycerol, betaine and the nu-
cleotide analogue 7-deaza dGTP did not improve the
efficiency of the PCR reaction.Better results were ob-
tained using an Advantage GC PCR polymerase mix
including 1 M of a commercial GC Melt Reagent
and 5% (v/v) DMSO together with a pre-PCR denat-
uration step of the DNA template. The introduction
of the respective mutation and the absence of nu-