<<

. 9
( 61 .)



>>

fragments of the virus genome, two methods can be employed: mechanical
(see Section 3.1 .I.) and enzymatic (see Section 3.1.2.) shearing. Shearing is
followed by further purification of DNA fragments wtth stzesbetween approx
33 and 40 kbp (see Notes 7 and 9) using centrifugation through a glycerol
density gradient (see Section 3.1.3.), blunting of the DNA fragments using T4
DNA polymerase (see Section 3.1.5.), and addition of synthetic linkers for clon-
54 de Wind, van Zul, and Berns

mg (see Section 3.1 S. and Note 10 for the criteria that the cloning linker should
meet; see also Note 11). This is followed (if necessary) by ligation to a modl-
fied cosmld vector (see Sections 3.1.6.-3.1.11. and Note 12), packaging in vitro
m phage lambda capslds, and mfectlon of E colz (the packaging and Infection
steps are done using a commercially available packaging kit). Resulting bacte-
rial colonies containing cloned virus DNA fragments are expanded, cosmlds
are isolated, and size, map location, and integrity of the inserts are analyzed by
restriction enzyme digestion and gel electrophoresis. Finally, a set of clones IS
selected m which the inserts cover all of the genomlc information of the virus,
and that have overlaps at their ends, as described above and depicted in Fig 2
(see Notes 13 and 14). After characterlzatlon by restrtctlon enzyme analysis,
the selected set of clones 1s assayed for the regeneration of viable virus by
overlap recombmatlon, after transfectlon of cultured cells. Resulting virus IS
analyzed by digestion of virus DNA followed by gel electrophoresls.
3.1.1 Mechanical Shearing of the Virus Genome
1 20 yg Purified virus DNA 1s taken up m 0.5 mL TE and loaded mto a 1-mL
syringe. Attach a 25-gage needle to the syringe, fix firmly, and squeeze into a
IO-mL polypropylene tube Repeat once
2. Transfer the sheared DNA to a rmcrofuge tube and preclpltate the DNA by the addition
of 0.1 vol3Msodmm acetate pH 5 2 and 2 vol ethanol MIX and leave on ice for 15 mm;
spm down for 5 mm m a mlcrofuge at maximum speed Wash the pellet m 1 mL 70%
ethanol and let the remaining ethanol evaporate at room temperature (see Note 15).
3. Add 100 pL TE to the pellet The DNA is dissolved by heating to 65™C for 5 mm,
followed by repeated plpetmg up and down or by overnight storage at 4°C The
DNA may subsequently be kept at 4°C
4 To check the sizes of the sheared fragments, 5 pL of DNA are loaded on a 0 5%
agarose mmigel Load 0 5 vg unsheared vu-us DNA as a control As marker frag-
ments, 0.5 pg uncleaved phage lambda DNA and 1 pg of a HzndIII digest of
lambda DNA are loaded (see Note 16) Run the gel until the Bromophenol blue
dye has migrated well off the gel. The DNA smear resulting from the shearing of
the virus genome should migrate well below the Intact virus genome, starting at
around the band of uncleaved lambda DNA and extending below that band.
5 Next, the DNA fragments ˜111 be size selected on a glycerol density gradlent
(Section 3.1 3.)
3.1.2. Enzymatic Shearing of the Virus Genome
1. Mix, m a mlcrofuge tube on ice, 40 pg virus genomlc DNA, 100 pL 10X DNase
buffer, dlstllled water to 1 mL. Add 10 ml of the DNaseI stock solution (DNaseI
end concentration: 2 5 pg/mL) Rapldly transfer the tube to a 15°C water bath
2 Take 250˜pL samples after 6, 9, 12, and 15 mm of mcubatlon. Immediately after
sampling, add to each sample an equal volume of phenol chloroform.lsoamyl-
alcohol and vortex gently (see Note 17) for 30 s to inactivate the enzyme
55
Characterization of a Herpesvws Genome
3. Spin all samples in a mrcrofuge at maximum speed for 2 mm. Remove the upper
phases each to a clean tube Add to each of these tubes 0 1 vol3Msodmm acetate
pH 5 2 and 2 vol ethanol Mix and leave on ice for 15 min, spm down for 5 mm m
a mtcrofuge at maximum speed. Wash the pellet in 1 mL 70% ethanol and let the
remaining ethanol evaporate at room temperature (see Note 15).
4 Add 50 FL TE to each pellet The DNA is dissolved by heatmg to 65°C for 5 mm,
followed by repeated up-and-down pipeting or by overnight storage at 4°C The
DNA can subsequently be kept at 4°C.
5 To check the sizes of the sheared fragments, 5 l.tL of DNA are loaded on a 0 5%
agarose minigel. Load 0 5 ng unsheared virus DNA as a control. As marker frag-
ments, 0.5 pg uncleaved phage lambda DNA and 1 pg of a UlndIII digest of
lambda DNA are loaded (see Note 16). Run the gel until the Bromophenol blue
dye has migrated well off the gel The DNA smear resultmg from the shearmg of
the vnus genome, should migrate well below the intact virus genome, starting at
around the band of uncleaved lambda DNA and extendmg below that band
6 Pool the fractions contammg sheared DNA of approximately the right size for
further fractionation on a glycerol density gradient (Section 3 1 3 )*
3. I. 3. Size Selection of Subgenomic Fragments by Centrifugation
Through a Glycerol Gradient
1. Using a gradient former, prepare m an SW40 tube two linear 1540% glycerol
density gradients, each of 11 mL m total
2. Carefully load the sheared DNA preparations, contammg approx 20 pg DNA
each m a volume of 100-200 pL TE buffer, on top of the gradients
3 Spin the tubes m an SW40 rotor for 5-7 h at 40,000 rpm, 2O™C.
4. After centrifuging, hermetically seal the tubes with parafilm and carefully punc-
ture the bottom of the tubes with a 25-gage needle. Take off the parafilm and
collect 12-drop (i e , 0 6 mL) fractions m prenumbered microfuge tubes.
5. Load 25-pL aliquots of every second fraction on a 0 5% agarose gel, using as
markers uncleaved lambda DNA and lambda DNA, cleaved with Hind111 (see
also Note 16)
6. Pool fractions contammg DNA from the desired size range, dilute wtth TE to 5
mL (dilute at least twofold), and centrifuge in a SW50 rotor for 5 h at 50,000
rpm, 20°C to prectpitate the DNA
7 Carefully discard the supernatant and drssolve the DNA m 20 pL TE.
8. Run 1 nL on a 0.5% agarose mimgel together with several loadmgs of between
0.1 and 1 pg of lambda DNA to estimate its concentration.
3.7.4. Addition of Synthetic Linkers to the Sheared DNA Fragments
and to the Cosmid Vector
The ends of the sheared- and size-selected vnus DNA fragments are blunted,
after which synthetic linkers are to be added at the ends for clomng (see Note
10). If this site is not also present in the cosmld, one of the unique clonmg sites
m the cosmld vector (most cosmld vectors contam, e.g., a unrque BumHI site)
de Wind, van Ziji, and Berns
56

must be converted to the new unique cloning site by linker addition, for which
we also provide the protocol. For both linker addition to the sheared virus DNA
fragments and to the cosmid vector, linkers should be phosphorylated at then
5™ ends, as also described in Sectron 3.1.5.
3. I .5. Blunting the Sheared Virus DNA Fragments
and Addition of a Restrict/on Site for Cloning
This subprotocol mcludes a number of separate enzymatic steps:
1. Blunting the sheared DNA fragments with T4 DNA polymerase (see Note 18),
2 Phosphorylation of the linker,
3 Ligation of an excess of lmker (see Note 19) contaming the clomng site, to both
ends of the DNA fragments,
4. Removal of the excess of lmkers by cleavmg wtth the restrictton enzyme for
which the lmker has a site followed by purrficatlon on an agarose gel.
Thts clomng procedure may partially be performed srmultaneously with the
addition of (the same) linker to the cosmrd vector (see Section 3.1.6.) It IS
recommended to set aside a sample of 0.1-0.2 pg after each step for analysis on
a 0.5-0.8% agarose mmlgel.
1 Mix on ice m a nncrofuge tube the following. 5 pg sheared DNA, 2 pL 1OX T4 poly-
merase buffer (seeNote 20), 1 pL of a 2-mA4 dNTP mtx, 1 U T4 polymerase, dtstrlled
water to 20 pL Incubate for 15 min in a 12 “C water bath and return the tube to Ice.
2. To mactlvate the enzyme, add to each tube 80 pL TE and 100 pL phenol:chloro-
form:isoamyl alcohol. Vortex for 30 s and centrifuge at maximum speed for 2
mm m a mtcrofuge Transfer the upper phases to clean mrcrofuge tubes, add to
each tube 0 1 vol3M sodium acetate pH 5.2 and 2 vol ethanol. MIX and leave on
me for 15 mm. Spm down for 5 mm m a microfuge at maximum speed. Wash the
pellet m 1 mL 70% ethanol and let the remaining ethanol evaporate at room tem-
perature (see Note 15). Dissolve the pellet in 5 VL TE by overnight storage at 4™C
or by mcubatton at 65°C for 15 mm
3. Mix in a microfuge tube: 1 pL 10X polynucleotide kinase buffer, 2 pg non-
phosphorylated linkers, 1 PL 10 mMATP, 10 U polynucleotide kmase, dlsttlled
water to 10 PL Incubate for 1 h at 37°C. Heat the tube at 65™C for 15 min to
inacttvate the enzyme.
4 Add to the microfuge tube containing the sheared and blunted virus DNA* a 50-fold
molar excess of phosphorylated linker (i.e., approx 7.5 ng), 2 pL ligation buffer,
5 (Weiss) Umts T4 ltgase, distilled water to 20 pL. Incubate overnight at 15°C.
5 Add to each tube 80 pL TE and 100 pL phenol chlorofonnlsoamyl alcohol, vortex
gently for 30 s, centrifuge for 2 min at maximum speed, and transfer the supematant to
a clean micromge tube. Subsequently, prectpnate the DNA by additton of 0 1 vol3M
sodium acetate pH 5 2 and 2 vol ethanol Store on Ice for 15 mm and centrtmge for 5
mm at maximum speed Wash the pellet with 1 mL 70% ethanol and dtssolve the DNA
m 10 pL TE by heating for 15 min at 65™C or by overnight incubation at 4°C.
57
Characterization of a Herpesvirus Genome
6. Add to the DNA: 5 pL of the appropriate 10X digestion buffer, 50 U of the restrtc-
tton enzyme for which the lmker has a site, and distilled water to 50 uL. Mtx well usmg
a micropipet and incubate for 4 h at the appropriate temperature for the enzyme.
7. Load the entire digestion mixture on a 0.5% preparative agarose gel and elec-
trophorese until the bromophenol blue marker has migrated 2 cm mto the gel.
8. Cut out the DNA smear and elute using an electroelutton apparatus.
9. After electroelutton, transfer the eluted DNA to a microfuge tube, add 1 vol
phenol:chloroform*tsoamyl alcohol, vortex for 30 s, centrifuge for 2 min at maxt-
mum speed, and transfer the supematant to a clean microfuge tube. Repeat tf neces-
sary. Subsequently, prectpttate the DNA by addition of 0.1 vol Na-acetate pH 5.2
and 2 vol ethanol Mix and store on ice for 15 min and centrifuge for 5 min at
maximum speed. Wash the pellet with 1 mL 70% ethanol and dissolve the DNA m
10 pL TE by heating for 15 mm at 65™C or by overnight mcubatton at 4°C.

3.1,6. Generation of a Cosmid Vector Containing
a Specified Unique Cloning Site by Linker Addition
to the addition of the cloning linker to the sheared and blunted
Similarly
vents DNA, the msertion of [the same] restriction site m the cosmid vector
consists of a number of separate enzymatic steps.
1 Cleavage of the cosmid vector to lmearity with the enzyme at whtch site a novel
cloning site wtll be generated;
2 Bluntmg of the lmearized cosmtd vector;
3. Ligatton of an excess of linker (see Note 19) contammg the clonmg site, to the
resulting molecule;
4. Removal of the excess of lmkers by cleaving wtth the restriction enzyme for
whtch the linker has a site followed by purificatton on an agarose gel. Steps 3 and
4 may be performed simultaneously with the additton of a linker to the sheared
DNA fragments as described m the previous section;
5. Religatton of the cosmtd that now contains a single linker molecule; and
6. Transformation of competent E ˜011. Bactertal colonies are expanded and DNA
of the lmker containing cosmid vector is tsolated and analyzed (see Note 2 1).
Set aside a sample of 0.1-0.2 pg after each step for analysis on a 0.5-0.8%
agarose minigel. In addition, It is recommended to prepare competent E coli
cells first (see Sectlon 3.1.7.).
I. Mix the following m a mtcrofuge tube: 5 l.tg covalently closed cosmtd vector,
4 FL of the appropriate 10X restriction enzyme digestron buffer, 20 U restrtctton
enzyme for which the site is to be sacrificed for the Insertion of a new clonmg
site, and distilled water to 40 pL. Incubate for 2 h at 37°C.
2. Put the tube containing the digest on ice and add 2 PL 2 mA4dNTP mix and 5 U
T4 polymerase (see Note 18). Incubate for 15 min m a 12°C water bath.
3. To inactivate the enzyme, add to the tube 60 pL TE and 100 PL phenol chloro-
fornmsoamyl alcohol. Vortex for 30 s and centrifuge at maxtmum speed for 2 mm
de Wind, van Zgl, and Berm
58
Transfer the upper phase to a clean microfuge tube, add 0 1 vol3Msodmm acetate
pH 5 2 and 3 vol of ethanol Mix and leave on ice for 15 mm Spm down for 10 mm
m a mlcrofuge at maxImum speed Wash the pellet m 1 mL 70% ethanol and let the
remaining ethanol evaporate at room temperature Dissolve the pellet m 10 PL TE
4. Mix m a mlcrotige tube: 2 pg lmeanzed and blunted cosmid vector with a 50-fold
molar excess of phosphorylated lmker (see Section 3.1 5 , step 3, I e , approx
200 ng), 2 pL llgatlon buffer, 5 (Weiss) units T4 llgase, and distilled water to
20 pL Incubate overnight at 15°C
5. Add to each tube 80 pL TE and 100 pL phenol:chloroform.lsoamyl alcohol. Vor-
tex for 30 s and centrifuge at maximum speed for 2 min in a mlcrofuge Transfer
the upper phases to clean mlcrofuge tubes, add to each tube 0 1 vol 3M sodmm
acetate pH 5.2 and 3 volumes ethanol Mix and leave on ice for 15 min Spm
down for 5 mm m a microfuge at maximum speed Wash the pellet m 1 mL 70%
ethanol and let the remaining ethanol evaporate at room temperature. Dissolve
the pellet in 10 pL TE The DNA may be stored at 4°C or at -20°C
6. Mix m a mlcrofuge tube 1 yg of the DNA, 5 pL of the appropriate 10X dIgestIon
buffer, 100 U of the restnctlon enzyme for which the lmker has a site, and dIstIlled
water to 50 pL Incubate for 4 h at the appropriate temperature for the enzyme.
7 Load the entire digestion mixture on a 0 8% preparative agarose gel and ele-
ctrophorese untd the dye band has migrated approx 2 cm.
8 Cut out the cosmid vector band and elute using a Geneclean (or slmllar) kit Take
the DNA up m 10 pL TE
9. Add to the DNA. 2 yL ligase buffer, 1 (Weiss) Unit T4 hgase, and distilled water
to 20 FL. Incubate for 2 h to overnight at 15°C The hgation mixture can be
stored at -2O™C (see Note 22).
10 Transform competent E. colz (see Section 3.1.7.) with 2 pL of the ligation mix-
ture, as described below (see Section 3.1.8.).
11 Inoculate the next day, using wooden toothpicks, 6-12 tubes contammg 2 mL LB
plus amplclllm with fresh single transformant colonies. Grow overnight at 37™C
m a shaking Incubator. Perform mnupreps as described in Sectlon 3.1.9
12. Test the quality of the competent cells by transformation of 0 1 ng supercooled
control plasmld (like pBR 322), as described in Section 3.1.8. The efficiency of
transformation should be equal to, or higher than, 5 x lo6 colomes per pg plasmld.
Also include, as a negative control, a mock transformation with no added DNA
13 Reinoculate a colony contammg the desired cosmld vector m a flask containing 15
mL LB + amplclllm, grow overnight m a shaking Incubator at 37”C, and isolate the
plasmld using a IO-fold scaled up version of the protocol described m Section 3.1.9.
3.1.7. Preparation of Competent E. coli Cells
Most protocols for making E. colr competent for uptake of DNA rely on an
incubation of cells m a solution containing CaCl,. The method described here,
acid trlturation (17),1s no exception to this and yielded good results m our hands.
We routinely use strain DH5; however, any strain may be suitable provided that
the strain 1s recombination deficient (recA-). Note: It 1s important that the glass-
59
Characterization of a HerpesvIrus Genome
ware used is thoroughly rmsed with stertle ultrapure water prior to autoclavmg.
In addition, all steps from step 4 on must be carrred out at 0-4OC.
1. Pick a fresh colony, inoculate a 200-mL sterile flask contammg 25 mL LB, and
mcubate overmght at 30°C m a shaking incubator
2 Inoculate a 2-L flask containing 500 mL LB with 5 mL cells of the overmght culture
3 Shake the culture at 30°C until the OD6a0 reaches 0 45-O 55
4. Transfer the cells to ice water and leave on ice for 2 h
5. Centrifuge the cells for 15 mm at 25OOg, 4°C
6. Gently resuspend the cells in 20 mL fresh ice-cold trnuration buffer and add,
subsequently, 480 mL more ice-cold trituration buffer
7 Incubate the cells on ice for 45 min
8. Centrifuge the cells for 20 mm at 18OOg, 4°C.
9. Gently resuspend the cells m 50 mL ice-cold trituratton buffer
10. While gently swtrlmg the cells m ice water, slowly add glycerol to a final con-
centration of 15% v/v
11 Ahqwot the cells m 0.2- to 1-mL portrons in mrcrofuge tubes and freeze on dry
ice The cells can be stored for at least 6 mo at -80°C
12. Test the quality of the competent cells by transformation of 0.1 ng supercoiled
control plasmid (hke pBR322), as described m Section 3.18 The efficiency of
transformation should be equal to, or higher than, 5 x lo6 colonies per pg plasmid
Also include, as a negative control, a mock transformation with no added DNA,
3.7.8. Transformation of E coli
1 Pipet l-10 pL of a solution contaming up to 50 ng DNA on the bottom of a sterile
mlcrofuge tube on ice (see Note 23)
2. Thaw, on ice, an ahquot of frozen competent cells and gently pipet 200 uL ice-
cold cells on the DNA solutton
3. Mix by tapping and Incubate for 3@-60 min on ice
4 Heat the tubes in a 42°C water bath for 90 s.
5 Transfer the tubes to ice water for 2 min.
6 Add 800 PL SOC medium and incubate at 37°C for 30-60 mm
7. Plate out ahquots on LB agar plates containing ampicillm (see Note 24)
8 Incubate overnight at 37°C.
3. I .9. Small-Scale Isolation of Plasmid DNA (˜mInipreps™
This procedure IS derived from the original alkalme lysis method (28). It can
easily be scaled up tf more DNA is required. In the latter case, use 15 or 50 mL
conical tubes instead of mmrofuge tubes and centrifuge 5X longer, at 5000g.
1 Inoculate numbered tubes with 2 mL LB containing ampicillm with fresh single
transformant colonies using wooden toothpicks. Grow overnight at 37°C m a
shaking incubator
2 Transfer 1 5 mL of the cultures to microfuge tubes and centrifuge for 1 min at
maximum speed

<<

. 9
( 61 .)



>>