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ity can be altered). Mutations can also be made that do not affect gene ex-
pression, but that otherwise affect the virus repltcatton cycle, such as the
alteration of DNA rephcatron orrgms, or of sequences necessary for pack-
aging herpes DNA mto virus particles. The functions of genes and other
DNA regions can also be studied by the swapping of DNA regions between
closely related viruses, by the replacement of genes with a marker (such as
ZacZ), or by the msertlon of an anttgemc pepttde tag mto a protein. Tags
can also be inserted mto a protein to enable mteractmg proteins to be copu-
rified and identified. Some mutations are more easily studied when cloned
into a plasmrd vector, from which a mutant protein can be expressed when
transfected into cultured cells, whereas other mutations require the mutant
sequences to be inserted mto the herpes genome.
Mutations can either be made by the addition or removal of large stretches
of sequence from the vu-us (usually accomphshed by the use of naturally
occurmg restriction sites), or by the msertton, deletion, or alteration of shorter
sequences (either by the insertion of “lmker” sequences into a natural restric-
tion site, “filling m” and religatron of a restriction site, or by the use of stte-
directed mutagenesis, where small, defined changes can be made).
Combinations of these techniques can allow most of the types of alteration
outlined above to be made.
2. Materials
1 50X TAE: 242 g/L Tris base, 57 1 mL/L glacial acetlc acid, 50 mM EDTA
2. 5X TBE. 54 g/L Tris base, 27.5 g/L boric acid, 10 mA4 EDTA
3 LB. tryptone 10 g/L, yeast extract 5 g/L, NaCl 10 g/L, pH 7 0 with 5M NaOH,
autoclave
4 LB agar. as above with 15 g/L agar
5 X-gal stock: 40 mg/mL X-gal in dlmethylformamlde.
6. T4 hgaseand buffer, restrrctlon enzymesandbuffers, T4 DNA polymerase,Tug
polymerase, and buffer by Promega (Madison, WI).
HSV Mutagenesis
3. Methods
All the methods described below require the region of interest wlthin
the herpes genome to have been previously cloned into a plasmid vector.
If the mutated sequence is to be studied m the context of the virus genome,
the region to be altered must also be flanked on either side by at least 1 kb
of unaltered sequence to allow efficient recombmatlon back mto the
herpes genome (methods covered elsewhere m this book). Some tech-
niques will require further subclonmg of only the sequence to be altered
mto a second plasmld vector, which can then be returned to the flanking
sequences after alteration for recombination mto the herpes genome.
3.1. Alterations Making Use of Restriction Sites
3.1.1. Initial Subcloning and Simple Additions or Deletions
Before herpes DNA sequences can be altered, the region of mterest often
needs to be subcloned from a larger parental plasmld, either to allow useful
restriction sites to be made use of, so that the DNA IS in a particular plasmid
vector (e.g., for the generation of single-stranded [ss] DNA for mutagenesis),
or merely so that the plasmld to be manipulated is of a more manageable size
General conslderatlons for subclonmg Include.
1. “Sticky”-end llgatmns are more efficient than blunt-ended hgations,
2 If possible, use a plasmld vector m whxh blue/white selection of recombinant
colonies is possible
3. Llgatlon together of DNA fragments whose two ends have each been cut with a
different restrlction enzyme are more efficient than where only one enzyme IS
used, smce in this case, only the desired recombinant can be produced (especially
useful where no blue/white selection 1s available) The use of two restrIctIon
enzymes also allows directional cloning.
4. The larger the DNA fragments to be hgated, the less efficient the subsequent
ligation
5. DNA of reasonable purity, e.g., purified by a commercial kit, such as the Wizard
system (Promega) or Qiagen (Hllden, Germany) systems, should be used.
6. Full sequence mformatlon/restnctlon maps should be avallable for all DNA frag-
ments/vectors used
Regions can be deleted from herpes DNA by simple dlgestlon with an
mdlvtdual or pair of restriction enzymes (sometlmes after blunt-ending of
the DNA) and rehgatlon, and sequences added by the same subclonmg pro-
cedures and with the same considerations as outlined above. The easiest way
to inactivate a particular herpes gene IS to insert a promoter/marker gene
cassette (such as SV40/LacZ from pCHlO1 [Pharmacla, Uppsala, Sweden])
into a unrque restrictlon site wlthm the plasmld-encoded gene, so that after
30 Coffin
cotransfectton with herpes DNA recombinant virus, plaques appear blue after
stammg with X-gal.
3.1.2. Subcloning Procedure
3 1 2 1 DIGESTION/BLUNT-ENDING OF DNA

Approximately 2-5 pg of each DNA (vector and insert) should be di-
gested with the appropriate restriction enzymes m a buffer optimal for
acttvtty (see manufacturer™s mstructions) in 100 pL using 220 U of each
enzyme. If two enzymes are bemg used, a buffer can usually be found in
which both enzymes are reasonably active. Incubate for I h at the appro-
priate temperature (usually 37°C). Do not use “One-Phor-All” or
“Multicore” type acetate based buffers if DNA IS to be subsequently
blunt-ended. If one DNA terminus needs to be blunt-ended (if DNA IS
being cut with two enzymes), digest DNA with the first restriction en-
zyme (site to be blunted) for 1 h, add I pL 25 mM each dNTP and ˜10 U
T4 DNA polymerase, and incubate at 37*C for a further 30 min (T4 DNA
polymerase works in most restriction enzyme buffers). Heat to 80°C for
20 mm (inactivates enzymes), cool to 37”C, add the second restriction
enzyme (site not to be blunted), and incubate for a further 1 h. If both
ends require blunt-ending add 1 pL 25 mM dNTPs and x10 U T4 DNA
polymerase after restriction enzyme digestion, and incubate at 37°C for
30 min. DNA should then be phenol-extracted and precipitated with etha-
nol (I/. DNA pellets should be washed with 70% ethanol by vortexmg
and recentrifugation before drying and resuspension m 10 pL of water.
3.1.2.2. PURIFICATION OF DNA

If the vector DNA has been cut with only a single enzyme, then no fur-
ther purificatton is required. If with two enzymes it, like the insert DNA,
must be separated from the unwanted DNA fragments m an agarose gel.
Cast a 1% LMP (LMP) agarose gel using the smallest wells available m 1X
TAE containing 0.4 pg/mL ethidmm bromide. Load the entire DNA sample
(10 pL), and run until the DNA fragments are well-separated from each
other as viewed by UV trans-illummatton. Excise the desired band, using a
scalpel, with the minimum possible excess surrounding agarose and mini-
mal exposure to UV.
3.1.2.3. LIGATION OF DNA
There 1sno need to purify DNA from the gel slice. Melted gel can be added
directly to the ligation mix Melt the gel slice(s) at 70°C for 5 min, vortex, and
add 1 or 2 PL of each melted agarose slice to a 5-PL ligation mix:
31
HS V Mutagenesis
One gel-purified Two gel-purified
Ligation using fragment fragments
c2 pL
Water 2 PL
Gel fragment (insert DNA) 2 PL 1 PL
Gel fragment (vector DNA) 1 PL
Vector DNA use various
amounts to
optimize hgation
(in water from Section 3.1.2.2.) OS-2 pL
5X T4 DNA ligase buffer 1 PL 1 PL
Total 5 PL 5 PL
Remelt agarose by brief mcubation at 70°C, and mixing, cool to room tem-
perature, add 1 pL T4 DNA ligase, mix, and mcubate at room temperature for
1 h. Add 5 pL of water, heat to 70°C to melt any agarose, mix, and add to com-
petent cells (see Section 3. I .2.4.)
Controls: (1) cut vector DNA, no ligase: checks effictency of vector
cut-should give no colonies, and (2) cut vector DNA + ligase: checks
efficiency of ligation-if cut with one enzyme should give many colo-
nies; if cut with two enzymes, should give many less colonies than when
insert DNA IS added.
3.1 2.4. TRANSFORMATIONOF E. coli
Competent cells are prepared by inoculating a single, freshly grown
colony of Escherzchia colz DH5 or similar into 100 mL of LB. This is
grown shaking at >200 rpm at 37°C for 2-4 h until swirlmg, opalescent
bacterial growth can just be seen. Cells are harvested by centrifugation at
3000 rpm for 10 min, resuspended in 20 mL ice-cold 100 m/t4 CaCl, re-
centrifuged, and finally resuspended m 4 mL 100 mA4 CaCl. These are
stored on me until used and may be kept for up to 4 d, or frozen m 200˜pL
aliquots at -70°C mdefmltely.
Ligated DNA from Section 3.1.2.3. above is added to 200 yL of com-
petent cells in a 15-mL plastic disposable tube, incubated on ice for 30
mm, transferred to a 42°C water bath for exactly 90 s, transferred back to
ice for 2 min, and 800 pL LB added. The tube is incubated by shaking at
37°C for 1 h before harvesting the cells by centrifugation as above,
resuspension m a small volume of the medium (2100 FL), plating onto
1% LB agar plates containing the appropriate anttbiotic (and X-gal [50
yglmL] if blue white selection is bemg used-IPTG is usually unneces-
sary), and incubated overmght at 37°C. Control: Transform with x10 ng
uncut vector DNA-checks competence of the cells.
Coffin
32

3.1.2.5. IDENTIFICATION OF RECOMBINANT COLONIES
Recombinant colonies are identified by plasmid mimpreparation and restrtc-
non digestion, If blue/whtte selection can be used, then white colonies are
picked. If a single fragment is being relegated or two fragments hgated m such
a way that self-ligation 1s not possible (i.e., two restriction enzymes used),
colonies can be picked at random. However, tf the vector is capable of self-
relegation m the absence of the inserted DNA (no blue/white selection), a
colony lift to transfer colonies to a nitrocellulose or nylon membrane must be
performed, followed by hybrtdizatlon with the insert DNA (radiolabel some of
the DNA m melted agarose from Section 3.1.2 2., e.g , Ready-To-Go kit from
Pharmacia) to rdenttfy recombinant clones. The manufacturer™s mstructions
for the membrane used (e.g., Amersham [Amersham, UK], HyBond N, or
HyBond N+) should be followed in this case.
Mmtprep of plasmid DNA: Single colonies identtfied as above are mocu-
lated mto 5 mL of LB and grown overnight m an orbital shaker (200 rpm) at
37OC.Followmg mcubation, transfer 1 5 mL of the culture to a mtcrofuge tube,
centrifuge m a mlcrocentrtfuge for 1 mm, and aspirate the supernatant. Next
sequentially add, and thoroughly mix by vortexmg, 100 pL of solution 1
(50 mA4 Trts-HCl, pH 7.5, 10 mM EDTA, 100 pg/mL RNase A), 200 PL of
solution 2 (0.2MNaOH, 1% Triton X-100), and 150 pL of solution 3 (3M KOAc
pH 4.8). Centrifuge for 3 mm, remove the pellet with a bent hypodermic needle,
add 500 pL of tsopropanol, mix, and recentrifuge for 5 mm. The pellet is then
washed with 70% ethanol, dried, and resuspended m 50 yL of water contammg
20 pg/mL RNase A.
Restrtction digestion of mmtprep DNA: Digest 3 nL of the mimprep DNA
from above with restriction enzymes (total Cl PL) suitable to distinguish
recombinant from nonrecombmant colonies in the appropriate buffer m a total
of 10 nL for 1 h. Directly run the 10 FL reaction mixture (+ 1 pL loading buffer
[50% glycerol, 1X TAE, 0.25% bromophenol blue]) on a 1% agarose gel.
A plasmid maxrprep (100 mL to 1 L culture) should then be performed,
either using a commerctally available kit (e.g., Qiagen tip-100 or 500) or by a
traditional method, such as cestum chloride density gradtent centrrfugatlon,
before, for example, cotransfection mto cells together with herpes DNA to gen-
erate a recombinant virus.
3.1.3. Filling in Restriction Sites
Subtle changes to a sequence (e.g., alteration of a promoter element or intro-
duction of a stop codon mto an open reading frame) can often be made by
“filling in” (5™ overhang) or “chewing back” (3™ overhang) of a restriction site.
In both cases, T4 DNA polymerase (which has both polymerase and exonu-
clease activity) can be used as m Sections 3 1.2.1.-3.1.2 5., above. The alteration
33
HS V Mutagenesis
m restrtctton pattern observed can then be used both to identify recombinant
plasmids and also recombinant viruses should the alterations need to be trans-
ferred to the herpes genome. A servesof truncated proteins can often be gener-
ated by cutting and filhng in of different restrrction satesas they occur along a
sequence. Inspection of the sequencesto be altered, and the sequencegenerated
after filling m will determine if the desired result can be achteved m this manner.
3.1.4. Oligonucleotide Insertion
Pairs of complementary ohgonucleotides, which when annealed leave pro-
truding “sticky” ends, can easily be inserted into a unique restriction site or
(preferably) between two different sites (since here only the desired recom-
binant can be generated) in a target sequence. These might encode an amino
acid sequence or a particular nucleic acid motif. Ideally, the inserted oligo-
nucleotide should contam a restrrctlon site to aid identtflcation of the desired
recombinant.
Method:
1. Digest target DNA with approprrate enzyme(s) and gel-purtfy as m Sectton 3 I 1
2 Set up two legation reactions, one containing ˜100 ng of each oltgonucleottde,
and one without (control), as in Section 3 1 1 Overlay wtth 50 uL mmeral 011 Do
not add hgase There is no need to phosphorylate the oligonucleotrdes
3 Heat reaction to 95™C, and cool slowly to room temperature in a large beaker
of water
4. Add hgase below the mineral oil, mtx, and mcubate at room temperature for 1 h
before adding the mrxture (below the mineral 011) to competent cells
5. Prck colonies, miniprep, and identrfy recombmants by the mtroductton of restrrc-
tion snes encoded by the oltgonucleotrdes or, If necessary, by a colony lift using
end-labeled oligonucleotide (see Section 3.2.) as a probe.
3.2. Linker insertion Mutagenesis (Scanning)
Linker scanning mutatagenesis (2) allows the fine mapping of regions
important for gene function (promoter or protein-coding regions), usually after
important regions have been initially defined at a more gross level by deletion
analysis. The general goal of a linker scanning procedure is to produce a series
of mutants in which overlappmg small sequences(˜10 bp) have been individu-
ally replaced by a common linker sequence along the length of the sequence
under study. Thus, although important amino acid or nucleic acid residues are
likely to have been replaced by the procedure, the spacing between promoter
elements or protein domains would remam unchanged.
The procedure requires four stages.
I Clonmg of the DNA of Interest mto a plasmrd vector.
ii. Generation of randomly spaced breaks m the sequence of Interest.
in. Deletion of a short sequence on each side of the breaks and insertion of hnkers.
IV Identification and characterlzatton of the mutants generated
A number of protocols for stages (11) and (in) have been devised, the major
stumblmg block being the generation of truly random breaks (by chemrcal
means) and the concurrent deletion of sequence together wtth the msertton of
the linker. Simpler procedures can be performed If the breaks m the sequence
are produced by partial digestion (to linearize) with frequently cuttmg restrtc-
tron enzymes (not allowing “full coverage” of a sequence) and if linker
sequences are then inserted without the concurrent deletion of a sequence of
equal length (precise spacing between sequencesis not maintained). Protocols
for this based on the method of De Wmd et al. (3) are described below.
1 Restriction digestion of target DNA Blunt-cuttmg restrictron enzymes with four-
base recogmtion sequences that cut with reasonable frequency m the DNA of
Interest should be used. Available enzymes are HaeIII, NZuIV, FnuDII (rso-
schlzomer BstUI), and AluI. Use of the restrrction enzymes at low concentration
allows the partial digestion of the DNA to give Imear fragments, whrch can be
purified on a polyacrylamrde gel
Set up five digestions/enzyme, 10 ug of DNA each, with 1,0.5,0 25,O 12, and
0 06 U of enzyme/l00 uL reaction, m the manufacturer™s recommended buffer
Inclusion of ethidnrm bromide at 50 ug/mL (FnuDII), 5 pg/mL (HueIII), or 0.5 pg/
mL (RsaI) increases the proportion of linear fragments (3,4) Incubate at 37°C for
15 mm, and transfer to an 80°C water bath for 20 mm to mactrvate the enzyme
2 Gel-purification. After partial digestion, phenol-extraction and ethanol precipita-
tion, plasmtd DNA that has been cut only once (linearized) is separated from
undrgested and multiply digested forms by separation on a 1X TBE-5% poly-
acrylamide gel (I) m comparison to plasmrd DNA that has been cut with an
enzyme that cuts only once. After electrophoresis, the gel is stained in ethidmm
bromide for half an hour (0.5 pg/mL m 1X TBE), viewed on a UV transtllumma-
tor, and the sample with the greatest proportion of linear DNA IS quickly excised
(in the smallest posstble gel fragment) with a scalpel DNA is extracted from the
gel by placing the excused fragment into the well of a 1% LMP agarose gel, run-
ning the DNA into the gel l-2 cm, and extracting the DNA from the gel using a
commercial kit or by meltmg and phenol-extraction. Resuspend m water at ˜200
ng/pL (estimated by agarose gel electrophoresis and comparison with DNA of
known concentratton)

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