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Nucleic Acids Research, Vol. 18, No 6
A rapid and simple method for introducing specific
mutations into any position of DNA leaving all other
positions unaltered
M.Tomic 13 , I.Sunjevaric1'3, E.S.Savtchenko1 and M.BIumenberg 12 *
Departments of Dermatology1 and Biochemistry2, N.Y U. Medical Center, 550 First Avenue, New
York, NY 10016, Permanent address- Institute Boris Kidric, Vinca, Yugoslavia
Submitted February 20, 1990
Recent developments in molecular biology focus on the
interaction of transcription factors and regulatory proteins with
their specific recognition sites in DNA. An essential component
of this analysis involves physical and functional interactions of
proteins with different DNA sequences. One of the experimental
approaches is to systematically mutagenize the target DNA, i.e.
to change the bases with which the proteins interact (1) Recently,
polymerase chain reaction (PCR) technology has been used to
introduce particular mutations into target DNAs (2) This
approach either depends on the adventitious position of a useful
restriction site, or introduces, besides the desired mutation,
additional changes in the DNA at the engineered restriction sites.
Our goal was to devise a simple method to introduce
predesigned point mutations, or a series of closely-spaced specific
mutations, into DNA segments that are too large to be directly
chemically synthesized (i.e. 0.3 to 3 kb), without changing a
single base pair other than the desired mutations. The method
is based on PCR and restriction enzymes that cut the DNA at
a short distance from their recognition sites. The sites for such
enzymes are amplified during the PCR, but are eliminated during
the subsequent restriction, ligation and cloning steps, thus
perfectly re-creating the target DNA except for the point mutation
designed into the experiment.
The experimental design uses four PCR primers as outlined
in Fig. 1. Two are on the outside of the region of interest and
could be kilobases away from the site to be mutated. They contain
restriction sites convenient for cloning, e.g. Hindin, EcoRI or
BamHI. Near the site to be mutated, two BspMI site-containing
PCR primers are synthesized so that after the digestion with
BspMI the BspMI sites are eliminated. The BspMI cuts with 4
bp, 3' extended cohesive ends and care should be taken that these
cohesive ends are both complementary to each other in the two
primers, and correspond to the template DNA, since these will
be retained in the final product. One or both of the two primers
contains the desired mismatches. These will result in mutations,
differences from the template DNA. The amplified DNAs are
digested with BspMI to remove the BspMI sites and expose the
cohesive ends. Digestion of the opposite ends with their cognate
restriction enzymes sets the stage for the three fragment ligationthe two BspMI cohesive ends, which are not self-complementary
but complementary to each other, are ligated to each other, and
the opposite ends are ligated into the cloning vector (Fig. 1).
This method can be used to introduce quickly and easily any
desired mutation within a given DNA segment. Except for the
* To whom correspondence should be addressed
desired changes, the DNA sequence remains unaltered. Besides
BspMI, additional enzymes that cut at some distance from their
recognition site could also be used. These are listed in Table 1.
Concerted simultaneous use of more than one such enzyme could
introduce mutation at several distant sites. We chose BspMI
because the cutting site is close to the recognition site, which
means the PCR primers can be shorter, because it has a 4 base
staggered cutting site that facilitates ligation and because it is
inexpensive. Using this procedure we have mutagenized, in the
human K# 14 keratin gene promoter, the sites regulated by the
nuclear receptors for retinoic acid and thyroid hormones.
REFERENCES
1 Myers.R M , Tilly.K and Maniatis.T (1986) Science 232, 613-618
2 Vallette.F , Mege.E , Reiss.A and Adesnik.M (1989) Nucl Acids Res
17, 723-733
TEMPLATE
DNA
AMPLIFY
I an
I
MIHIWIII *j«-i
UOAII INTO T-
T-CUT VtOTOM
Figure 1. Experimental design Below the template DNA the structures of the
four primers are indicated The open boxes contain recognition sites for enzymes
' 1 ' , '2' and BspMI The vertical open arrows mark the cutting site for the BspMI
The short diagnonals represent the few additional nucleotides necessary for the
efficient cutting by the restriction enzymes The horizontal arrows contain
sequences complementary to the template Asterisks mark the positions of the
speciftcially introduced mismatches that result in the product that contains
mutations
Table 1. Restriction enzymes that cut at a distance from their recognition site
Enzyme
Alwl
Bbvl
BbvII
BspMI
Fold
Gsul
Hgal
HphI
MboII
Mnll
Piel
SfaNI
Taqll
TthlllH
Recognition Site
GGATC
GCAGC
GAAGAC
ACCTGC
GGATG
CTGGAG
GACGC
GGTGA
GAAGA
CCTC
GAGTC
GCATC
GACCGA
CAA(A/G)CA
Cutting Site
N4/N5
N8/N12
N2/N6
N4/N8
N9/N13
N167N14
N5/N10
N8/N7
N8/N7
N7/N7
N4/N5
N5/N9
N11/N9
N11/N9
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