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3. Linker msertlon The linker used should be 12 bases in length for optimal liga-
tion efficiency and to keep the protein m frame, and palmdromic (1.e , self-
complementary) to allow double-stranded insertion into the digested plasmtd after
annealing has occurred. The linker should also contam a restriction site for an
enzyme that does not cut in the plasmtd undergoing mutation to allow easy char-
acterization of mutants containing the inserted linker The sequence used should
also not contam any translational stop stgnals (TGA, TAG, TAA) or allow the
generation of such stop signals after insertion For this purpose, the inserted
35
HS V Mutagenesis
sequence can start wrth a Y-C The use of phosphorylated lmkers Increases the
efficiency of insertlon
Linker phosphorylatlon.
Ohgonucleotlde 500 ng
10X kinase buffer
2 5 pL
(200 mivl Tns, pH 7 6, 100 mA4 MgCl,)
IO n&˜ATP 2.5 yL
T4 DNA polymerase 5U
Incubate at 37°C for 30 mm and use directly m the ligation reactlon below
Set up three 5-pL ligation reactions for each eluted linearized DNA band using
10,30, and 100 ng of linker and 1 PL (S-100 ng) of purified linearized DNA
Purified DNA ™ PL
5X T4 DNA ligase buffer 1 PL
Phosphorylated linker XPL
Water to4pL
T4 DNA llgase ™ PL
After llgatlon, transform into competent cells, and perform colony lifts onto,
e g., HyBond-N (Amersham) followmg the manufacturer™s mstructlons Trans-
ferred colonies are then probed with y-32P-1abeled linker (using T4 polynucle-
otlde kmase* mix 5 pCl [Y-˜˜P]ATP, 5 ng linker, 4 pL 10X kmase buffer [above],
water to 40 yL, 10 U T4 polynucleotide kmase, and incubate at 37°C for 1 h) and
linker contammg colonies are Identified by autoradlography Posltlve colonies
are mmlprepped, and their structures are analyzed by restriction analysis (using
the restriction site introduced with the linker), and finally by DNA sequencmg
By this means, many mutants may be identified with near-random insertion of
the linker sequence to allow regions of fimctlonal significance to be probed, either
while still m plasmid form or after transfer to the viral genome
3.3. Site-Directed Mufagenesis
SpecGk, small changes to a sequence are made by site-directed mutagenests
usmg specific mutagenic oligonucleotides and target DNAs either cloned into
an M 13 or phagemld vector, or into any or a more specialized plasmid for the
newer PCR or “repalr oligonucleotide” methods. These can Include introduc-
tion of a restriction site, alteration of individual amino acids in a protein
sequence, mtroductlon of a stop codon, or alteration of particular promoter
elements (to name but a few). However, m each case, the general conslderatlons
for all site-directed mutagenesisprocedures and the design of the ohgonucleotlde
to be used are the same whichever method IS being used:
1. Small mutations(l-3 bases)aremore efficiently generatedthan large mutations
(5+ bases).
Coffin
36
2. Replacement of nucleotides is much more efficient than deletion or insertion of
nucleotides.
3. Highly pure ohgonucleotides should be used (HPLC or “cartridge” purified)
4 Longer oligonucleotides generally give a higher efficiency of mutagenesis than
do shorter ones Optimal size IS obvtously dependent on the number of mls-
matches In general, 2&25 mers can be used to replace l-3 bases m the center of
a sequence, and for larger replacements/deletions/msertions (up to x10 bases),
ohgonucleotides with ˜15 unchanged bases on either stde of the bases to be
changed can be used with reasonable efficiency
5 Palmdromic oligonucleotide sequences should be avoided, as should “unusual”
lookmg sequences sequences that look truly random are best and should opti-
mally have a G + C content of z 60%
6 Use of an oligonucleotide that mtroduces a restriction site (if possible) greatly
eases the identification and characterization of potenttal mutants
The orrgmal and most widely used methods for We-drrected mutagenests
devrsed by Kunkel et al (.5j, usmg uracrl containing DNA and mismatch repair
were relatively complex systems and, although often rehable, were time-con-
suming. Individual users would usually have to optimize the various stepsbefore
successful mutagenesis. However, newer and simpler methods have now been
described that are both highly reliable and widely used. These are either based
on the use of an ohgonucleotrde to remove a unique restriction site (or similar
alteration to repair an antibiotic reststance gene) or on PCR, and since these
provide many advantages over the older methods, these will be described here.
3.3.1. Unique Site Ehmination-Based Mutagenesis
Advantages:
Rapid, easy, and efficient
Any plasmid vector can be used.
Kits commercially available
Multiple mutations can be made concurrently.
Multiple rounds of mutagenesis can easily be performed.
A number of commercially available kits are available for unique site ehmi-
nation-based mutagenesis (e.g., “U.S.E.” from Pharmacia; “Chameleon” from
Stratagene [La Jolla, CA]), all based on the same origmal system of Deng and
Nickeloff (6), a modification of which will be described here. This method,
like the Kunkel method outlined above, relies on a mutagenic oligonucleotide
annealing to the plasmid DNA (preferably ssDNA generated by supermfectron
with bacteriophage, but alkali-denatured dsDNA can be used with lower effi-
ciency), and the use of T4 DNA polymerase/T4 DNA hgase to synthesize and
repan the remainder of the complementary DNA strand. However, mutated plas-
mid is here selected for by the use of a second ohgonucleotide, which deletes a
HSV Mutagenesis

unique restriction site, and thus after digestion with this restrtction enzyme and
transformation, will only allow growth of bacteria containing plasmld m which
the site has been deleted. Experiment has shown that a very high proportion of
plasmid in which the restrictton site deletion has taken place (up to 95%) will
also have mcorporated the mutagenic oligonucleotide. Multiple mutagenic oh-
gonucleotrdes can be used successfully at one time (up to 4) and further rounds
of mutagenesis performed by the sequential use of different unique restriction
sites m the plasmid vector. A slmtlar method is available from Promega
(“Altered Sites”) m which, instead of deletion of a unique restriction sate, an
Inactive antlblotlc resistance gene is repaired, and thus only plasmid m which
repair has occurred will allow growth in the presence of that anttbiotrc. However,
here the DNA to be mutagemzed must be cloned mto a speciahzed plasmld vec-
tor (purchased from Promega), adding another stage to the mutagenesis process.
Method: For best results, ssDNA should be produced by supermfectlon of
phagemid containing bacteria with helper-phage, although alkali-denatured
plasmid DNA can be used with lower efficiency, and both the mutagemc and
restrrctron site ehmmatlon oligonucleotide should be phosphorylated (Section
3.2.). Twenty-base olrgonucleotldes removing any unique site m the vector to
be used (without interrupting an open reading frame) can easily be designed,
and rf these also Introduce a new unique site, multiple rounds of mutagenesis
can easrly be performed Ohgonucleotrdes for use m pUC- and pBR-based vec-
tors are available from Pharmacia and Statagene for this purpose. The repau-
deficient E. colz strain BMH71-18 mutS should also be purchased, e.g., from
Promega, which prevents repair of the mutations after the mutagenesis reac-
tton and before final transformatron into a standard lab E colz strain.
1 Preparation of ssDNA
a. Pick a single freshly grown colony into 5 mL of selectrve media, add =I O™O
PFU M13K07, and grow overmght at 37°C shakmg vigorously. (Stock
M13K07 should be bought and prepared according to the manufacturer™s
instructions, e g , Promega, or by standard methods [I] >
b. Pellet the cells (6OOOg,4°C 10 min), and decant the supematant to a fresh tube
Add 900 pL 20% PEG6000,2 SMNaCl, mix, and incubate at room tempera-
c
ture for 30 mm to precipitate phage.
d. Pellet phage at 12,OOOg, 4™C; 20 mm, and decant superanatant (white pellet
should be visible), aspirate to remove traces of PEG
e. Resuspend in 500 nL water containing 100 pg/mL RNase A, and transfer to a
microfuge tube
f. Phenol/chloroform-extract at least three times, each time vortexmg for at least 30 s
g. Add 0.5 vol of 7 5Mammonium acetate and 3 vol of ethanol, and vortex. Spin
in a microfuge for 10 mm, discard the supematant, and wash wrth 70% etha-
nol by vortexing and respmning before air-drymg the pellet and resuspension
in 20 pL of water; 5 pL should grve a bright band on an agarose gel.
Coffin
38
2 Mutagenests reaction and restriction digestion
a. Anneal. Mix 100 ng ssDNA, 2.5 ng restrtction site ehmmation ohgo, 1 25 ng
mutagenic ohgo, 2 pL 10X annealing buffer (200 mA4 Tris-HCI, pH 7 5,
100 n&f MgCl,, 500 mMNaCl), and water to 20 pL
Heat to 70°C for 5 mm, cool slowly to room temperature, and place on ice
b Synthesis* Add to the annealmg reaction 3 pL 1OX synthesis buffer (100 rnM
Tris-HCl, pH 7.5, 5 mM each dNTP, 10 mM ATP, 20 m&J DTT), 10 U T4
DNA polymerase, 2 U T4 DNA hgase, and water to 10 pL (final annealing/
synthesis reaction vol 30 uL)
Incubate at 37°C for 90 mm Incubate at 80°C for 20 mm Add 10 U of the restric-
tion enzyme at the site that IS being deleted Incubate at 37™C for 1 h
3. Transformation mto E colz BMH7 1- 18 m&S
a. Prepare competent BMH7 l-1 8 mu& cells as m Section 3.1 1.
b Add the entire synthesis reactton to 200 uL competent cells, incubate on ice
for 30 mm, heat shock at 42°C for 90 s, and add 5 mL LB before mcubation at
37°C for 1 h with vigorous shaking Add ampicillm to a final concentration of
100 pg/mL, and continue mcubation overnight
4. Final selection and and transformation mto standard host E co/z
a Mimprep 1 5 mL of the culture from step 3, and resuspend in 85 pL of water
contammg 5 pg/mL RNase A Add 10 pL of the optimal 10X buffer for
the enzyme site being deleted and 5 pL (50 U) of the appropriate restriction
enzyme. Incubate at 37™C for 1 h, add 300 pL of water, and phenol-extract before
ethanol precipitation
b Resuspend the precipitated DNA in 10 uL of water, and transform mto 200 pL
of competent cells (standard lab stram, e.g , DH5a, JM 10 1) as in Section 3 1 1
Spread ˜/lo and “/lo onto amplcrllm plates, and incubate at 37°C overnight
Colonies should be checked for the mutation by mmiprep and restriction dtges-
tion, and finally by sequencing
3.3.2. PCR- Based Methods
Advantages:
DNA in any plasmid vector can be used
Easy and efficient
Ktts available
Disadvantage:
Since PCR itself can Introduce mutations, the enttre cloned DNA should be
sequenced after the procedure to check for unwanted mutattons.
A number of different PCR-based mutagenesis procedures have been devel-
oped, and a method will be described here that has been used many times and
with high effictency m our laboratory to introduce single-point mutations,
multiple-point mutations, random mutations (at defined sites) using degener-
US V Mutagenesis 39

plasmid-speclhc primer
gene-specific mutant primer
Insert
\ m2
I


ml
PCR w&h primers
pl andml


PCR with primers
pZ and m2
4.2
\/
A mutatedbases
/
t
overlappmg
sequence - =


B
\ “=



\/
Gel purify and rnlx products A and B,
PCR with primers pl and p2.
(Overlapping sequences allow annealing
of products A and B before PCR with
primers pl and p2)




clone products


i

Fig. 1. Outline of the scheme for mutagenesis by overlap extension.

ate primers, insertions, and deletions. The method is based on the site-directed
mutagenesis by overlap extension procedure described by Ho et al. (71, which
requires two plasmid-specific primers, allowing amplification of the entire
insert sequence (universal sequencing primers can often be used), and two
complementary gene-specific mutant primers.
The method is outlined in Fig. 1, where two initial PCR reactions are per-
formed using in each case a plasmid-specific primer (pl or p2) and a gene-
specific mutant primer (ml or m2) to generate two mutated PCR products,
which overlap at one end. After gel purification, and mixing, this overlap allows
Coffin
40

annealing of the two products before a second amplification step with the two
plasmtd-specific primers alone to generate a full-length mutated sequence that
can be cloned mto the plasmtd of chorce.
Gene-specific mutant pruners: The design of gene-spectficpnmers 1southned in
Ftg. 2 and should generally, for samplepomt mutations, be at least25 baseslong. As
with site-directedmutagenesis, greater numbersof mismatchesrequtre longer oligo-
nucleottdes. Random mutattons can be made at defined sites tf degenerate ohgo-
nucleotides are used (i.e., where all four basesA, T, C, G, or parttcular mixtures are
included at defined positions within the ohgonucleotide) Self-complementartty
withm pruners, “unusual” sequences (runs of mdlvtdual nucleotides or repeatedele-
ments) and particularly high or low G + C content should be avoided.
Plasmrd-specific primers: The plasmrd-spectfic primers can m many cases
be the M 13 forward and reverse universal sequencing primers, which have the
advantage that the polylmker of the plasmrd m which the sequence has been
cloned will also be amphfied during the second PCR, and can thus be used for
digesting and cloning the final PCR product. If these primers are unsuitable,
primers containing suttable restriction sites (recognmon site starting at least 5
bases from the 5™ end to allow efficient digestion) should be designed. How-
ever, even without restrrction sites, the final PCR product can easily be cloned
into commerctally available plasmtds lmearized to include an overhanging 3™-
T (e.g., Promega T-vector system). This system takes advantage of the over-
hanging 3™-A Taq polymerase added to all PCR products during ampltficatton.
Method:
1. Set up two PCR reacttonscontaming template DNA and either primers pl and
ml or p2 and m2 We routinely use Tag polymeraseand buffer from Promega
10 pL 10X buffer, 7 PL 25 mMMgCl,, 1 pL 10 mkIdNTPs, 300 ng each pl +
ml or p2 + m2, ˜50 ng template, and water to 100 pL Overlay wtth 100 pL of
mineral oil
2. Denature DNA by heating to 98°C for 5 mm in a thermal cycler, remove, add
50 U Tag polymerase below the mineral oil, and mtx by ptpettmg.
3 Cycle 20-30 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 2 mm.
4 Run 10 pL of each reaction on a 1% LMP gel, and excise the appropnate bands m the

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