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Nucleic Acids Research, 1994, Vol. 22, No. 5
© 1993 Oxford University Press
A new method for strand discrimination in sequencedirected mutagenesis
Haruo Ohmori
Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan
Received January 4, 1994; Accepted January 26, 1994
Sequence-directed mutagenesis is an essential approach for
studying the roles of critical residues in determined sequences
of DNA and protein. A wide variety of methods have been
developed, among which the standard ones with a single-stranded
DNA as the template for mutagenesis (1—5) include the following
steps: i) synthesis of an oligonucleotide (oligomer) containing
a desired base-substitution(s), ii) phosphorylation of the oligomer
at the 5'-end by T4 polynucleotide kinase and its annealing to
the single-stranded template DNA, iii) in vitro synthesis of the
complementary strand and sealing to make the closed circular
heteroduplex, and iv) transformation of the heteroduplex into
E.coli cells. The yield of mutants should be theoretically 50%,
but it is much lower because, for example, the mutagenic primer
is displaced in the in vitro reaction or the mismatch is repaired
in the transformed bacterial cells. To increase the yield of the
intended mutation, it becomes important how to discriminate the
mutated strand from the nonmutated parental strand. In the
method developed by Kunkel (1), the template DNA is prepared
from dut ung double mutant bacterial strains in which dU is
incorporated into DNA in place of T at some proportions, and
the parental strand is preferentially degraded upon transformation
into wild-type (i.e., ung+) cells. In other methods (2, 3), the in
vitro DNA synthesis is carried out with nucleotide analogues that
render the synthesized strand resistant to digestion by some
restriction enzymes, and the parental strand is degraded and
resynthesized in a series of enzymatic treatments before
transformation. Other methods (4, 5) make use of enzymatic
linkage of the mutagenic oligomer with another oligomer which
either corrects a nonsense mutation in the drug resistance gene,
or eliminates a unique restriction site, on the vector. They require
two rounds of transformation to select the desired mutant. Here
I describe a new simple method for the strand discrimination,
which is based on the unique property of replication of
ColEl-type plasmids.
Replication of ColEl and related plasmids depends on synthesis
of an RNA (designated RNA II) transcribed around the origin
and its hybridization to the template DNA strand (6). RNA II
serves as the primer for DNA polymerase I after being cleaved
by RNase HI when the enzyme is present. In the absence of the
enzyme, RNA II remains hybridized to the template DNA strand
and thereby displaces the non-template strand, on which the first
DNA synthesis takes place. Various alterations at the ori site (the
transition point from the primer RNA to DNA chain) or small
deletions around the HaeU site within the region coding for RNA
II abolish the replication activity in wild-type strains with active
RNase HI, but they give little effects onreplicationin rnhA strains
lacking the enzyme (7, 8). A 2-bp deletion at the HaeU site (5'-AGCGCC-3' to 5'-AGCC-3') was introduced by the Kunkel
method into the phagemid vectors pTZ18U and pTZ19U
(obtained from Bio-Rad) to construct pTZ18Urrh and
pTZ19Urrh, respectively. The derivatives are usually propagated
in AK101(9), an mhAv.cat derivative of JM101, and they can
transform JM101 at efficiency of less than 10~4 in comparison
with AK101. Insertion of a DNA fragment for mutagenesis at
the multiple cloning sites can be detected by loss of the lacZa
complementation activity, that is, formation of white-colored
colony on X-gal plates, when AK101 is used as the host for
transformation. For mutagenesis, a mutagenic primer is linked
during the in vitro DNA synthesis and sealing reaction with a
correction (selection) primer to restore the replication activity
in rnhA+ cells. After transformation of the resultant
heteroduplex into the rnhA+ cells, the first round replication
yields the mutant progeny (A in Fig. 1) and the non-mutant
progeny (B). The mutant progeny continues replicating, but the
Table 1. Result of mutagenesis
Exp 1
Exp 2
Eagl
a)
b)
c)
d)
5
3
0
6
9/14
6/14
V139M
G295D
6/14
5/14
a)
b)
c)
d)
In both experiments the reactions contained the selection pnmer [5'-CGGGAAGCGTGGCGCI 1 lCTCATAG-3'1 (the template DNA lacked the 2 bases complementary
to those underlined). In addition to it, Exp 1 contained the mutagenic primers pBgin [5'-TGTGATGCCAG(a)ATCTTTTCCA-3'] and pEagl [5:-CACGTTCGGCC(g)GTAGAGA-3'], and Exp 2 contained pV139M [5'-ACAAAGACAAC(t)AT(c)AGCTTGAATA-3'] and pG295D [5'-TAACAACGTTG(a)I(c)CGTACAAATC-3']
(the base underlined was substituted from the one indicated by a lowercase letter in the parenthesis). The sign + or - denotes the presence or absence, respectively,
of the substitution. The numbers of the clone containing the substitution are indicated on the right, and the ratios of the substitution by each oligomer are presented
on the last line.
Nucleic Acids Research, 1994, Vol. 22, No. 5 885
vector
In vitro DNA synthesis
transformation
In vivo replication
YES
NO
This mutagenesis method is very simple and rapid, since the
non-mutant progeny is selected out during replication in the
transformed cells. It requires neither a series of in vitro enzymatic
treatments to degrade the template strand (2, 3) nor two rounds
of transformation (4, 5). Unlike in the case of the Kunkel method,
the efficiency of mutagenesis by this method is not disturbed even
if RNAs contaminated in the template DNA preparations and they
primed the complementary strand synthesis, since the specific
sequence must be corrected to give rise to transformants. Also,
it is unlikely that the final products may contain unexpected DNA
alterations or rearrangements resulted from degradation of the
template DNA. The yield of mutation may be improved by
increasing the relative ratio of mutagenic primer versus correcting
primer and also by using a mismatch repair deficient strain (for
example, a mutS mutant) for transformation. It should be also
possible to apply this method to double-stranded DNAs with slight
modifications of the conditions in the annealing and synthesis
reaction.
Replication In rnh*ce\\»
ACKNOWLEDGEMENT
Figure 1. Principle of the method. The three arrowheads (a, b and c) indicate
synthetic oligomers for mutagenesis, in which a is the selection pnmer to correct
the deletion at the HaeU site (indicated by the filled circle), and b and c are
mutagenic primers.
non-mutant progeny is unable to replicate because the RNA II
with the 2-bp deletion, after cleaved by RNase HI, cannot be
utilized as the primer for DNA polymerase I (6).
In the following, I describe one example of the mutagenesis
using this method. A 1450 bp fragment coding for the actin gene
(ACT1) of Schizosaccharomyces pombe (10) was inserted into
the vector pTZ19Urrh to construct pSPACTl. Single-stranded
DNA of pSPACTl was prepared after infection with M13KO7
helper phage (11). The annealing mixture in a 10 ft\ [20 mM
Tris (pH7.5), 2 mM MgCl2, 50 mM NaCl] contained the
template DNA (about 0.5 pmole) and the three 5'-phosphorylated
oligomer (about 6 pmole each): one (25 mer, a in Fig. 1) to
correct the replication defect, and the other two (21-23 mer,
b, c in Fig. 1) to make mutations within the ACT1 gene. In one
experiment (Exp 1) the two mutagenic oligomers contained a
single base substitution to introduce a new restriction site {BgHl
or Eagl), and in another experiment (Exp 2) the oligomers
contained two base substitutions to introduce an amino-acid
change. The mixture was heated for 10 min at 65°C and slowly
cooled down to room temperature. After additions of 1 /tl of 1 OX
buffer [5 mM each of 4 dXTPs, 10 mM ATP, 100 mM Tris
(pH7.9), 50 mM MgCl2, 20 mM DTT], 0.5 /xl of T4 DNA
polymerase (1 unit, from Bio-Rad) and 1 y\ of T4 DNA ligase
(3 unit, from Bio-Rad), the mixture was incubated at 37°C for
90 min for the complementary strand synthesis and sealing
reaction. An aliquot (5 /tl) was used to transform MV1190 (11),
a recA derivative of JM101, to ampicillin resistance. About
100-fold more transformants were obtained with the above
reaction products than with the product of the control reaction
without any primer. 14 clones of the transformants, each from
the two experiments, were examined for the presence of mutation
either by restriction enzyme analysis (Exp 1) or by DNA
sequencing (Exp 2). The results shown in Table I indicate that
the yields of mutations within the ACT1 gene were around 50%
(9, 6, 6, and 5 out of 14).
I thank Dr D.Gallwitz for providing me with pSPA2 DNA.
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