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microfuge at 10,OOOg for 2 mm
3 Resuspend the pellet m absolute ethanol and repeat this step twice.
359
Analysis of HSV-DNA and RNA
4. Dry the pellet, resuspend m 100 pL of DNA extraction buffer, incubate at 37°C
for l-12 h, boil for 15 min, and take 10 uL for PCR

3.2. Extraction Procedure for RNA
1. Transfer tissue scraped from the slide to a sterile Eppendorf tube containing 0 8
mL TRI and homogenize usmg a Polytron homogemzer.
2 Store homogenized samples for 5 min at room temperature
3 After addition of 0.2 mL of chloroform, vortex samples for 15 s, and store for
2-3 mm at room temperature.
4. Centrifuge samples in a microfuge for 15 mm. RNA is found m the upper phase
5 Precipitate RNA from the upper aqueous phase with 0 5 mL of isopropanol for 10
mm at room temperature. Microcentrifuge samples for 10 mm at room temperature
6. Wash the RNA pellet with 70% ethanol and allow to air-dry.
7. Dissolve extracted RNA m 50 pL distilled water.

3.3. Amplification of DNA
Amplify lo-p,L samples of extract in a final volume of 100-pL m PCR assay
buffer for 30 cycles, at 95°C for 15 s, 54°C for 15 s, and 71°C for 1.5 mtn.
Amplified gB DNA IS 191 bp long, using the gB gene primers described above.
3.4. Construction of Competitor gB DNA
1. A mutant 19 1 bp competitor gB PCR fragment can be constructed, with a HpaII
restriction site 29 nucleotides from the 3™ end, by using the wild-type gB
PCR product as template for amplification with the wild-type gB 5™ primer
and a new 3™ primer (5™ AGA-AAG-CGC-CCA-TTG-GCC-AGG-TAG-
TAC-TCC-GGC-TG3™) m which nucleotide 29 IS changed from G to C, mtro-
ducmg a HpaII sate internal to the ortgtnal 3™ primer. The ampltftcatton
conditions are as described earlier
2. Gel purify the mutant gB product and quantify by optical density

3.5. Competitive Quantitative DNA-PCR
The amount of viral DNA in the extracts is first approximated by titration
against a tenfold dilution series of the mutant gB fragment, followed by a more
precise quantitation by titrating the extracts against a 2-fold dilution series
spanning the first determination.
1. Add 10 uL of extract to samples of a tenfold dilution series of the mutant com-
petitor gB fragment ranging from 100 pg to 1 fg.
2. Amplify the mixtures for 30 cycles using the original primer pair as described
3. Dilute reaction products 200-fold and amplify again for two cycles with radioac-
tive tracer in a 20-uL final volume of PCR Assay Buffer, to eliminate heterodimer
formation (13).
4. Add 20 U of HpaII and incubate the mixture at 37OC overnight.
5. Electrophorese the entire volume on a 4% NuSieve GTG agarose gel m 1X TBE
Ramakrishnan, Fink, and Levine
360

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WILD TYPE
i -191 bp
MUTANT -162tip
/



2 DAY 7 DAY 8 WEEK

Fig. 1. Competitive Quantitative DNA-PCR of gB. DNA from 2 d, 7 d, and 8 wk hip-
pocampal extracts were coamplified with a twofold dilution series of competitor mutant
gB DNA using gB primers as described in the text. The radioactive amplified PCR prod-
ucts were digested with &“a11 and electrophoresed on 4% NuSieve agarose gels. Gels
were dried and exposed to X-ray film. A representative autoradiograph is shown here. (2 d)
Lane 1, 100 fg of mutant gB DNA coamplified with an uninfected hippocampal extract;
lanes 2-7, twofold dilution series of competitor mutant gB DNA ranging from 100 fg (lane
2) to 3.1 fg (lane 7), coamplified with infected trigeminal extracts. (7 d and 8 wk) Lane 1,
100 fg of mutant gB DNA coamplified with an uninfected hippocampal extract; lanes 2-6,
twofold dilution series of competitor mutant gB DNA ranging from 100 fg (lane 2) to 6.25
fg (lane 6), coamplified with infected trigeminal extracts.

buffer. Dry the gel and expose to X-ray film (Hyperfilm-MP, Amersham, Arling-
ton Heights, IL) at -70°C.
6. Visual comparisons of the wild-type and mutant bands allow for a rough approxi-
mation of the amount of viral DNA in the extract.
7. Add 10 & of extract to an appropriate twofold dilution series of mutant competi-
tor gB fragments, usually from 100 to 3.1 fg.
8. Repeat steps 2-5. A representative autoradiograph is shown in Fig. 1.
9. Count the amount of radioactivity in each band using a system like the AMBIS
Radioanalytic Imaging System.
10. Prepare standard linear regression curves by plotting net counts per minute for
each sample in the dilution series against the amount of input mutant gB DNA in
the series. A software program like the Graph PAD INPLOT software program
(Graph PAD Software, San Diego, CA) may be used for this. Using the regres-
sion coefficient obtained from the standard curve and the net cpm from the wild-
type bands, the point of equivalence is determined and the amount of gB DNA in
the original extracts calculated.
3.6. Amplification of RNA
RNA templates are amplified using a combined RT-DNA amplification
method (9,24) in a single tube, using a single buffer system.
1. Treat extracts with DNase I (1000 U) for 60 min at 37°C to destroy the DNA template.
2. Incubate extracts in a final volume of 100 mL of RT-PCR assay buffer, at 37°C
for 30 min to permit reverse transcription.
361
Analysis of HSV-DNA and RNA

73 PROMOTER 6 PRIMER (IA™IJ
5™ CC-TCG-AAA-TI™A-ACC-CTC-ACT-AAA GAC-AGC-AAA-CCC-GTC-AG 3™
lkQdk-C!4C!-?ld˜
“l3Rlrx3E”

Fig 2. Schematic of the use of a 12-bp bridge oligonucleotide to stabilize the T3
RNA polymerase promoter and the 5™ LAT primer for ligation

3. Amphfy using LAT primers at 95°C for 1 mm and 60°C for 1 min for 30 cycles,
followed by a single extension at 72°C for 10 min. This results m a 195-bp prod-
uct with a BsaHI restriction enzyme site located 2 1 nucleotides from the 3™ end
3.7. Construction of Competjtor LAT-RNA
A mutant LAT-PCR product can be constructed in which the cytosine at resi-
due 22 from the 3™ end 1s converted to thymidine, resulting in the loss of the
BsaHI site. An oligonucleotide codmg for the T3 RNA polymerase promoter (5™
GCT-CGA-AAT-TAA-CCC-TCA-CTA-AA 3™) 1s ligated to the 5™ end of the
LAT 5™ primer (5™ GAC-AGC-AAA-CCC-GTC-AG 3™) using a 12-bp brtdge oli-
gonucleotide (5™ TGA-TTT-CTG-TCG 3™) complementary to SIXbasesof the T3
promoter oligonucleotide and SIX bases of the LAT 5™ primer as shown in Fig. 2.
1 Treat 5 pg of LAT 5™ primer with 10 U of T4 polynucleotide kinase Extract the
reaction products with phenol-chloroform, precipitate in ethyl alcohol, dry, and
resuspend in 10 pL autoclaved distilled water
2. Ligation of the T3 promoter oligonucleotide and the kmased LAT 5™ ohgonucle-
otide m the presence of the bridging oligonucleotide (5 pg each) IS accomplished
using 30 U of T4 DNA hgase m a final volume of 30 uL for 12 h at 14°C.
3. The T3 promoter-5™ LAT ohgonucleotide ligation product (5 JJL of the ligation
reaction mixture) is used m combmation with the 3™ LAT mutant primer (5™ ACG-
AGG-GAA-AAC-AAT-AAG-GGA-EGC-C-3™) to amplify the wild-type LAT
fragment creating a T3 promoter-mutant LAT DNA fragment that has lost the
BsaHI site 22 nucleotides from the 3™ end of the PCR product.
4. Electrophorese PCRproductson a 1% low-melt agarose 1X TBE buffer, ex-
the in
cise the corresponding band and confirm the presence of the mutation by BsaHI di-
gestion. The wild-type 3™ LAT primer anneals downstream to the point of mutation,
permitting the amplification of the mutant LAT using the original LAT primer pair.
5. Confirm thenatureof theT3-mutantLAT construct PCRamplification
by usingtheT3
ohgonucleottde as the 5™ primer and the 3™ LAT primer and the original primer pan.
Both primer sets should amplify a mutant LAT fragment that has lost the BsaHI site.
6. Subject 5 uL of the T3-mutant LAT DNA template to in vitro transcription using a T3
RNA polymerase kit (Promega, Madison, WI) in a final volume of 200 pL at 37OC
min according to the directions of the manufacturer After 60 mm of incubation, add
20 more units of RNA polymerase and continue the reaction for an additional 60 mm
Ramakrishnan, Fink, and Levine
362

12345678
12345678
12345678
MUTANT -195 bp
WILD TYPE 174 op



7 DAY 8 WEEK
2 DAY
Fig. 3. Competitive Quantitative RT-PCR of LAT-RNA. RNA from 2 d, 7 d, and 8
wk infected hippocampal extracts were,reverse transcribed and coamplified with a two-
fold dilution series of competitor mutant LAT-RNA using LAT primers as described in
the text. Radioactive RT-PCR products were digested with BsaHI and electrophoresed
on 4% NuSieve agarose gels. Gels were dried and exposed to X-ray film. A representa-
tive autoradiograph is shown. (2 and 7 d) Lane 1, RT-PCR products from infected
hippocampal extract after RNase treatment; lanes 2-6, RT-PCR products from a twofold
dilution series of competitor LAT-RNA, ranging from 50 fg (lane 2) to 3.1 fg (lane 6),
added to infected hippocampal extracts; lane 7, RT-PCR products from infected hippoc-
ampal extract; lane 8, RT-PCR products from infected hippocampal extract without
addition of reverse transcriptase. (8 wk) Lanes 1-5, RT-PCR products from a twofold
dilution series of mutant LAT-RNA, ranging from 50 fg (lane 2) to 3.1 fg (lane 6), added
to infected hippocampal extracts; lane 6, RT-PCR products from infected hippocampal
extract; lane 7, RT-PCR products from infected hippocampal extract after RNase treat-
ment; lane 8, RT-PCR products from infected hippocampal extract without addition of
reverse transcriptase.

7. Digest the DNA template with 10 U of RNase-free DNase, for 60 min at 37°C.
Extract the reaction products with phenol-chloroform, precipitate with ethyl
alcohol, dry, and resuspend in 100 pL of autoclaved distilled water. Quantitate
RNA by measuring optical density.
3.8. Competitive Quantitative RT-PCR
1. To obtain a first approximation of the amount of LAT-RNA in the brain extracts,
5 pL of extract is mixed with a IO-fold dilution series of mutant LAT-RNA rang-
ing from 1 pg to 1 fg and subjected to RT-PCR as described.
2. Amplify the samples for 30 cycles, dilute 200-fold, and following reamplification
for an additional two cycles, electrophorese the entire reaction mixtures in 4%
NuSieve GTG agarose gels, dry the gels, and expose to X-ray film, from which
visual estimates of RNA quantities can be made.
3. To arrive at more accurate determinations of the amount of LAT-RNA in the ex-
tracts, coamplify 5-a samples with an appropriate twofold dilution series of com-
petitor mutant LAT-RNA ranging from 50 to 3.1 fg, using the same procedures as
described above.
4. Process the RT-PCR products, quantify radioactivity using the AMBIS system
and analyze counts by linear regression as described for the viral DNA determi-
nations. A representative autoradiograph is shown in Fig. 3.
Analysis of HSV-DNA and RNA 363

3.9. In Situ PCR
1. Latently infected animals are perfused with 4% paraformaldehyde, the ganglia
embedded m paraffin, and cut mto 6-p sections on a mlcrotome
2. Sections of infected trigeminal ganglia on glass slides are deparaffinized m xylene
(3 x 2 min)
3 Sections are successively rehydrated with graded ethanols m the following order:
100% ethanol (2 x 2 mm), 70% ethanol (2 min), and 50% ethanol (2 mm)
4. Wash sections in PBS (pH 7.5) for 5 min, twice.
5 Sections are then treated with 1% HC 1 m PBS for 5 mm and then washed m PBS
(3 x 5 min).
6. The sections are rinsed m PCR Buffer II (3 x 5 min)
7. 25 PL ofln situ PCR reaction mixture is layered onto the sections, which are each
covered with a glass cover slip and sealed using nail polish, taking care to ensure
that the polish does not seep into the reaction mix.
8. PCR amplification 1s carried out in a BloOven II Thermal Cycler (BioTherm,
Fairfax, VA) m two stages, first for 3 cycles at 92°C for 1 mm, 54°C for 30 s,
and 72™C for 30 s, followed by 25 cycles at 92™C for 15 s, 54™C for 15 s, and
72°C for 15 s.
9 After amplification, the cover slips are removed and the sections washed succes-
sively with 1X SSC (2 x 5 mm), 50% formamide in 1X SSC (3 x 15 min, 56”C),
and 1X SSC (2 x 15 mm)
10. Following a rinse in Trls-buffered saline (pH 7 5) and 5% normal goat
serum, the dlgoxlgenln-labeled amplified DNA 1s treated with an antl-
dlgoxlgenm antibody conjugated to alkaline phosphatase (1:250 Boehrmger
Mannheim, Mannhelm, Germany), and visualized with BCIP/NBT (Vector,
Burlmgame, CA)
11. Color development 1s monitored visually, and stopped typically after approx 30
min, by washing with 0. 1M Tris-HC 1, pH 7 5, 1 mM EDTA. Typical positive
neurons are shown in Fig. 4A

3.70. Controls for the In Situ PCR
A number of controls are required to determine that the zn situ PCR results
are specific for the HSV- 1 gB gene sequence.
1. In sztu PCR 1s carried out using gB primers and uninfected ganglia to show
absence of any labeled nuclei (Fig 4B)
2. Using a primer pair for the HIV Tut gene not present m the tissue, no 1˜1 sztu
signals should be detected m infected ganglia (Fig. 4C)
3. DNase treatment of sections from infected ganglia prior to PCR should com-
pletely eliminate the PCR signals (Fig. 4D)
4. Finally, DNA extracted from sections of infected ganglia after PCR 1scompleted
should hybridize to gB specific probes in Southern blot analyses. Smgle radloac-
tive bands of the expected length should be observed (Fig 5)
Ramakrishnan, Fink, and Levine
364




Fig. 4.1˜ Situ PCR of HSV- 1 DNA in Trigeminal Ganglia. (A) In situ PCR of a
6-p infected trigeminal ganglionic sections with oligonucleotide primers for HSV- 1
gL3DNA sequences and digoxigenin-labeled nucleotides at 8 wk postinnoculation. (B)
Control, showing uninfected ganglion amplified in situ with HSV-1 gB primers. (C)
Control, showing 8-wk postinoculation ganglion amplified in situ with primers for
HIV tat. (D) Control, showing 8-wk postinoculation ganglion, amplified in situ with
primers for HSV- 1 gB gene after overnight DNase-1 treatment. Bar = 100 u.


4. Notes
1. PCR: A number of factors can contribute to nonspecific amplification. These can
include:
a. Contamination of PCR reagents: Use the best quality of reagents possible,
and use them exclusively for PCR. We routinely aliquot out all our solu-
tions and freeze them away at -20°C. We also use aerosol-resistant tips
for pipeting to minimize crosscontamination. Designate one area in the
lab exclusively for PCR work. When possible, DNA and RNA samples are
prepared in separate rooms. In addition, it is absolutely essential to run suit-
able negative controls (i.e., without target sequence) each time an experi-
mental PCR is performed.
b. Titrate the 7™aq polymerase with the specific target. Too much enzyme can
result in nonspecific background.
c. Titrate primers from 50 ng to 1 yg.
Analysis of HSV-DNA and RNA 365

123




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( 61 .)



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