Silver staining of nucleic acids. Applications in virus research and in diagnostic virology

Journal of Virological Methods, 7 (1983) 185-198

185

Elsevier

SILVER STAINING RESEARCH

J. LINDSAY

OF NUCLEIC

ACIDS.

AND IN DIAGNOSTIC

WHITTON,

FIONA

APPLICATIONS

IN VIRUS

VIROLOGY

HUNDLEY,

BARBARA

O’DONNELL

and ULRICH

DESSELBERGER* Institute of Virology, University of Glasgow. and MRC Virology Unit, Church Street. Glasgow Gl I5JR. U.K. (Accepted

21 June

The rapid visualise

1983)

and sensitive

double-stranded

method

of silver staining

(ds) and single-stranded

silver ions to nucleic acids in gels is stoichiometric staining

can be applied

silver staining

to problems

DNA

RNA

acids in polyacrylamide

and independent

in virus research

viral genome

of nucleic

(ss) molecules

of the GC content

and to the analysis

analysis

gels is used to

of both DNA and RNA. The binding

diagnostic

of clinical

of

of ds DNA. Silver virus isolates.

virology

INTRODUCTION

Silver staining of gels containing proteins (Merril et al., 1979; Oakley et al., 1980; Merril et al., 1981; Morrissey, 1981; Sammons et al., 1981; Dubrayand Bezard, 1982), lipopolysaccharides (Tsai and Frasch, 1982) or nucleic acids (Somerville and Wang, 1981; Berry and Samuel, 1982; Herring et al., 1982; Follett and Desselberger, 1983) has recently been used in increasing frequency because the procedure is rapid and very sensitive. The present paper shows wider application of silver staining to nucleic acid analysis

in basic and diagnostic

virology.

Double-stranded

(ds) and single-stranded

(ss) molecules of both RNA and DNA. extracted from viruses and electrophoresed on polyacrylamide gels can be visualised to a similar degree of sensitivity. Evidence on the stoichiometry MATERIALS

of silver binding

to nucleic

acids is presented.

AND METHODS

Materials

Acrylamide, N,N’-methylenebisacrylamide and N,N,N,N’,-tetramethylethylenediamine were purchased from Bio-Rad; ultrapure urea was from Schwartz-Mann

*To whom correspondence

016609.34/83/$03.00

should

be addressed

0 1983 Elsevier Science Publishers

B.V.

and

186

ethidium bromide reagent grade. Preparation

was from

BDH

Chemicals.

All other

chemicals

used were of

of nucleic acids

ssRNAs

Human influenza viruses of types A and B were grown in embryonated eggs or on MDCK (canine kidney) cells, purified by ultracentrifugation through sucrose gradients (or through 30% sucrose cushions) and the RNA extracted as described previously (Palese and Schulman, 1976). The maximum amount from virus of one egg was approximately 1 pg. Virus harvested yielded less RNA which was not quantitated.

of RNA obtained per one Petri dish

dsRNAs

Rotavirus RNAs were prepared directly from human isolates (Follett and Desselberger, 1983). In most cases less than 1 pg of RNA was analysed; O.D. measurements of RNA extracts from human isolates were too low to allow accurate quantitation. ssDNAs 0X174

virion

ssDNA

was purchased

from New England

Biolabs.

dsDNAs

Herpes simplex virus type 1 (HSV-1 strain 17) and type 2 (HSV-2 strain HG52) and HSV isolates from patients were grown on confluent BHK 21 Cl3 cells and the DNA extracted from the supernatant of infected cultures (Hirt, 1967). The patients’isolates were passaged 2-4 times before use and titres of 10’ to lo* PFUs/ml DNA equivalent to 3 X 10’ to 3 X lo* PFUs of virus was analysed. quantitated after extraction. BamHI

fragments

of highly purified

HSV-2 (strain

were obtained. DNA was not

HG52) DNA were cloned into

the BamHI site of pAT153 and propagated in Escherichia coli K12 strain HBlOl as described (Davison and Wilkie, 1981). Adenoviruses were grown on 293 cells (Takiff et al., 1981) in single wells (20 mm diameter) of Linbro plates and the DNA extracted as described by Wade11 and de Jong (1980). A maximum of lo8 TCID,, of virus/Linbro well was obtained. No quantitation of extracted DNA was carried out. Fragments generated by Hae III digestion of oX174RF DNA were purchased from New England Biolabs. Restriction

endonucleases

Restriction endonucleases were purchased from Bethesda and used as described (Maniatis et al., 1982).

Research

Laboratories

187

Gel electrophoresis Electrophoresis of rotavirus and of influenza virus RNAs was carried out on 2.8% polyacrylamide 6 M urea slab gels (25 X 15 X 0.15 cm) using Loening’s buffer system (Palese and Schulman, 1976) at 150 V and 50-60 mA overnight. The same gel system was used to separate restriction endonuclease fragments of HSV DNAs and of adenovirus DNAs, and to electrophorese 0x174 virion DNA. The oX174RF DNA fragments and digests of cloned HSV-2 DNA fragments were subjected to electrophoresis at 70 V and 10 mA overnight on 5% polyacrylamide slab gels (25 X 15 X 0.15 cm) using ‘Tris-borate-EDTA (TBE) buffer (Maniatis et al., 1982). Silver staining Gels were stained

with silver nitrate

(pH 5) as originally

described

by Sammons

et

al. (1981) and as applied by Follett and Desselberger (1983). The only modification was termination of the reducing step by soaking the gels in 5% acetic acid (Herring et al., 1982). Gels were photographed using transmitted light and Ilford FP4 film. Ethidium

bromide staining

Gels were stained

in ethidium

bromide

(4 ug/ml)

in 1 X TBE.

Densitometry Ilford FP4 negatives were printed onto transparent bands measured using a Joyce Loebl Microdensitometer were calculated

by cutting

out peaks from photocopies

weighing them; the weights were converted the photocopy paper as reference. RESULTS

AND

film and the absorption of 3CS. The areas under peaks of densitometer

tracings

and

into areas using the weight of 100 mm2 of

DISCUSSION

dsRNAs Silver staining of the dsRNA segments of rotaviruses has recently been used to monitor the emergence of new strains in the human population (Follett and Desselberger, 1983) during outbreaks of infantile gastroenteritis and also to analyse rotaviruses isolated from calves (Herring et al., 1982). The sensitivity of silver staining of rotavirus RNA was found to be 0.3 to 0.4ng/segment (Herringet al., 1982). Similarly, subnanogram amounts of reovirus genomic dsRNA have been detected in polyacrylamide gels by silver staining (Berry and Samuel, 1982). In order to study quantitative aspects of silver ion binding to nucleic acids (Jensen

188

Fig. 1. Panel A: RNA of three independent separated

on a 2.8% polyacrylamide

described

in Materials

pattern

and methods.

Print made on transparent

2; that in lane 3 is of RNA pattern

gel indicate A. Numbers

the genomic under

segments.

peaks indicate

isolates of human

rotavirus

6 M urea slab gel. RNA preparation,

RNA segments.

and Desselberger,

electrophoresis

1983),

and staining

as

film. The RNAs in lanes 1 and 2are of RNA

1 (Follett and Desselberger.

Panel B: Densitometer

(Follett

1983). The numbers

curves of lanes 1-3 (indicated

to the right of the to the left)of panel

189

and Davidson, rotavirus transparent

RNA

1966) in polyacrylamide were silver stained

gels, gels containing and photographed.

film (Fig. IA) and densitometricaliy

evaluated

the separated Prints

segments

of

were then made on

(Fig. IB). Plots of the areas

under peaks of a densitometer tracing against the molecular weights of the RNA segments (Espejo et al., 1980) gave straight lines (Fig. 2) indicating a stoichiometric reaction of the rotavirus RNA with silver ions. This assumes that the RNA segments occur in equimolar amounts in the rotavirus virion as they do in reovirus particles (Shatkin et al., 1968). The fact that the straight lines of plots in Fig. 2 (and in Fig. 4B and 58, see below} cannot be extrapolated through zero is ascribed to background staining which occurred to variable degrees and increased with the amount of nucleic acid applied to a slot. When individual slots (width 5 mm) were loaded with more than 1 ug of rotavirus RNA (bovine rotavirus), the densitometry of each silver-stained segment gave a uniform maximum peak, showing that even the low molecular weight segments bound silver to an extent indistinguishable by densitometric analysis from that of the high molecular weight bands (unpubl. results).

moletulllr

might

(dalton.)

Fig. 2. Piot of areas under against the molecular (o), and lane 3 (0)

peaks of densitometer

weight (daltons)

ofsegments

tracings of rotavirus

(Fig. 18) of human

rotavirus

RNA. Values obtained

RNAs (Fig. 1A)

from lane

I (a),lane 2

P

191

ssRN.4~ Figure 3 shows the characteristic migration patterns on a polyacrylamide RNAs extracted from three influenza A and three influenza B virus strains.

gel of the Each track

was loaded with the RNA extracted from virus obtained from one infected egg (a maximum of 1 ug of RNA; tracks l-6) or from one 50 mm dish of infected MDCK cells (tracks 7-12). By using recent reference strains as standards, the procedure seems to be well suited to the rapid monitoring of both major and minor changes in the RNA migration patterns of new influenza virus isolates. A minimum of approximately 2 ng/RNA segment of influenza virus could be detected (results not shown), i.e. the procedure is slightly less sensitive than for dsRNAs (see above). When influenza virus RNA which had been labelled with 32P in infected MDCK cells (Palese and Schulman, 1976) was silver stained after separation on a polyacrylamide gel and then autoradiographed for several days, it was found that autoradiography was about ten times more sensitive than silver staining (results not shown). Silver staining has been successfully applied to monitor large scale purification of viroid RNA (D. Riesner, pers. comm.), a single-stranded covalently closed circular RNA with a high degree of internal base-pairing. ssDNi4s Samples containing different amounts of 0X174 ssDNA were run on a polyacrylamide gel and silver stained. A single band was found. The lowest detectable amount was approximately

2 ng (results

not shown).

dsDN.4s of dsDNA

Two sets of serial dilutions HaelII) were electrophoresed

Fig. 3. RNA from influenza slab gel. RNA preparation, was prepared

(oX174RF

type A and type B viruses was separated electrophoresis

from harvests

fragments

on the same 5% polyacrylamide

and staining

as described

DNA digested

with

gel. One group

was

on a 2.8% polyacrylamide under Materials

before virus inoculation,

1and 7, RNA of influenza virus A/PR/8/34 (H3N2); lanes 3 and 9, RNA ofinfluenza of Virology, influenza

Glasgow);

virus B/Hong

lanes 4 and 10, RNA of influenza Kong/8/73;

lanes 6and

changed

B/Maryland/57

in later isolates

strain

4 and

virus B/Maryland/57; of influenza

10). Migration

(lanes 2,3,8.9 and 5,6,11 and

vit’us A/Texas/l/77

(H3N2) (isolate 2974, January

12, RNA ofinfluenza

RNA segments

(lanes

to

seeded with 2 X IO6

lanes 7-12) was applied to each slot ofthe gel. Lanes

(H IN 1); lanes 2 and 8, RNA of influenza

virus A/Glasgow/82

to the right of lanes 1,4,7 and 10 indicate of influenza

RNA

of three eggs or three Petri dishes of MDCK cells and the RNA equivalent

virus grown in one egg (lanes 1-6) or in MDCK cells of one Petri dish (50 mm diameter, cells and grown to confluency

6 M urea

and methods.

12).

virus B/England/4/82. A/PR/8/34

1982, Institute

lanes 5 and

I I, RNA of The numbers

strain (lanes 1 and 7) and

of corresponding

RNA segments

has

12

3

6

5

4

7

8

9

lo

1353 1078 872

B area

under

wak

(mm’)

t 2oo

1

I

I

I

I

I

I

,

,

I

1

2

3

4

5

6

7

6

Q

b 10

molecular

mi6ht

(dalton8)

I 10-S

193

silver stained

and the other stained with ethidium

that bands representing detected ethidium

approximately

bromide,

The result (Fig, 4A)shows

0.5 ng of DNA (closed circles in Fig. 4A) were

by silver staining whereas the minimum amount of DNA detected by bromide staining was approximately 5 ng (open circles Fig. 4A). Therefore,

the sensitivity of silver staining of dsDNA was approximately ten times greater than that of ethidium bromide staining. The silver stained gel was subjected to densitometric analysis and Fig. 4B shows plots of areas under DNA peaks against their molecular weights. The straight lines obtained confirm the stoichiometric natureofthe reaction between silver ions and dsDNA. Jensen and Davidson (1966) had found that the strength of binding of silver ions to DNA in solution increased with increasing GC content at pH 5.6. It was of interest to establish whether or not this observation applied to the silver staining of DNA in gels. Hence, a dsDNA of known sequence (IL. Whitton et al., in prep.) was digested, yielding fragments of widely differing GC content. Figure 5A shows the silver stained gel and densitometric tracing of an AvaI digest of pBz (the BamHI z fragment of HSV-2 (strain HG52) cloned into pAT153). Figure 5B illustrates stoichiometric binding of silver ions by these DNA fragments in spite of the wide variation in GC content. The difference between this result and that of Jensen and Davidson (1966) may be due to the fact that the concentration of silver ions used to stain nucleic acids in gels is at least IOO~fold greater than that at which the differential effect of GC content on silver ion binding in solution was observed. Potential applications of silver staining to nucleic acid analysis in molecular biology are widespread. It allows the construction of detailed restriction maps of DNA fragments using ten-fold less DNA and restriction enzyme than DNA staining with ethidium bromide requires (L. Whitton, in prep.). Silver staining avoids two disadvantages associated with the use of nick-translated DNA for mapping purposes. Firstly, nick-translated

DNA

must be labelled

uniformly

and remain

sufficiently

intact

to

yield defined fragments after digestion. Secondly, nick-translated DNA fragments cannot be analysed on denaturing gels and therefore may not be sized accurately. Furthermore silver staining can be used to detect DNA:RNA hybrids such as those generated

by nuclease

ping (L. Whitton,

Fig. 4. Panel A: oX174RF silver stained; were: Tracks

DNA digested

tracks 6-10 stained

separated

bromide.

tracings

the number

gel. Tracks

1-5

of DNA applied to each track

3 and 8.40 ng; tracks 4 and 9, 20 ng; tracks 5 and fragment

in each track isindicated (not shown) against

of base pairs in each fragment

I (a), lane 2 (o), and lane 3 (0).

map-

outweighs

as deduced

from the DNA

to theleft ofeach bromidestained

the molecular

track by gel. Panel

weight (daltons)

for lanes I, 2 and 3 of the silver stained gel in Panel A. The molecular

by multiplying from lane

on a 5% polyacrylamide

gel and by open circles (0) on the ethidium

B: Plot of areas under peaks of densitometer

mRNA

of silver staining

The total amounts

on the left show the length in base pairs ofeach

(Sanger et al., 1978). The limit of detectability

the DNA fragments obtained

with IIaeIII,

with ethidium

closed circles (0) on the silver stained

calculated

1977), and hence facilitate

1 and 6, 160 ng; tracks 2 and 7, 80 ng; tracks

IO, 10 ng. The numbers sequence

Sl (Berk and Sharp,

in prep.). The high degree of sensitivity

(Panel A) by the factor

of

weights were 660. Values

194

A

1

L

18.1

9.1

4.4

3.2

23

1.3

1.1

0.99

54

63

80

72

73

63

67

60

mol~~lar (dsltonr)

wel9ht x IO-5

percentage GC content

2

4

6

8

10

12

14

16

18

20

molecutar wat9llt fdaltonr) x m-5

195

the fact that the staining bromide.

Silver staining

procedure

takes slightly

longer than staining

with ~thidium

of nucleic acids in gels is not suitable for preparative

purposes

as silver-stained nucleic acids cannot be eluted from gel slices (unpubl. results). Genomic analyses of clinical DNA virus isolates can be quickly performed using the silver staining procedure. HSV isolates from patients (identified as such by reacting reference HSV antisera with infected cells in an indirect immunofluorescence test) together with a reference strain of HSV 1 (strain 17) and a reference strain of HSV 2 (strain HG52) were grown and the DNAs extracted. Aliquots of DNA were then digested with different restriction enzymes and the fragments separated on polyacrylamide gels and silver stained. A representative result is shown in Fig. 6. All the clinical isolates were HSV-l and showed characteristic individual patterns deviating from that of the HSV-I reference strain and from each other (Lonsdale, 1979; Buchman et al., 1980). Adenovirus replicated in 293 cells of one Linbro plate well yielded enough DNA to be analysed in a silver stained polyacrylamide gel. Bgl II digests of adenovirus reference strains (of serotypes 2,5 and 7) are shownin Fig. 7. Digests of Hirt extracts (Wade11 and de Jong, 1980) from infected cells were compared with digests of DNA obtained from gradient-purified virions. The method is currently being used to investigate the genomes of clinical adenovirus isolates obtained during a local outbreak of keratoconjunctivitis (B. O’Donnell, unpubl. results). ACKNOWLEDGEMENTS

We thank Dr. Andrew Davison for discussion and for initially supplying us with a BamHI digest of DNA from purified HSV-1 and HSV-2, Dr. Vivien Mautner for advice and for providing us with DNA from purified adenovirus of different serotypes, and Dr. J. Barklie Clements JLW was supported by a Medical

for critical reading of the manuscript. Research Council Training Fellowship

Fig. 5 Panel A: The lower part shows a lane of a 5% polyacrylamide have been separated

fragments

cloned into the BarnHI

fragmsent are shown. The densitometer peaks

of densitometer

calculated

by multiplying

tracing

gel (run from left to right) on which

from an Ava I digest of pBz (the BamHI z fragment

site of pAT153).

The molecular tracing

in Fig. 5A against

the number

weight (daltons)

is displayed

G84/785.

of HSV 2 (strain HG52)

and the GC content

(%) of each

above the gel lane. Panel B: Plot of areas under

the molecular

of base pairs in each fragment

weights

(daltons)

(Whitton

of the fragments,

et al.. in prep.)

by 660.

196

A 1

E3 1

23456789

Fig. 6. BamHI

digests of HSV DNA separated

and silver staining

as described

Tracks

1-7, independently

(strain

17) reference

1, HSV 2 (strain experiments.

strains. HG52);

in Materials

obtained

on a 2.8% polyacrylamide

and methods.

6 M urea slab gel. Electrophoresis

Panel A: Hirt extracts (Hirt, 1967) of HSV DNA.

clinical HSV isolates; track 8, HSV 2 (strain HG52), and track 9. HSV I

Panel B: HSV reference track

2

2. HSV

I (strain

strains purified

and extracted

17). The gels in panels

as described

(16). Track

A and B are from different

197

Fig. 7. Hgl II digests of adenovirus

DNAs of serotypes

2 (lanes 5 and 6), 5 (lanes 3 and 4) and 7 (lanes

1and

2). DNA extracts

from infected cells (Wade11 and de Jon& 1980; lanes I, 3 and 5) and from purified virions 6 M urea siab gel. (lanes 2, 4 and 6: 0.2 pip of DNA was used) were separated on a 2.8% polyacrytamide Electrophoresis represent

residual

and silver staining partially

digested

as described

in Materials

DNA fragments.

and methods.

The faint bands

in track

6

198

REFERENCES

Berk, A.J. and P.A. Sharp,

1977, Cell 12, 721-732.

Berry, M.J. and C.E. Samuel, Buchman,

T.G., T. Simpson,

1982, Anal. Biochem. C. Nosal, B. Roizman

124, 180-184. and A. Nahmias,

1980, Ann. N.Y. Acad. Sci. 354,279-

290. Davison,

A.J. and N.M. Wilkie,

Dubray,

G. and G. Bezard,

Espejo,

R., E. Martinez,

Follett,

E.A.C.

Herring,

1981, J. Gen. Viral. 5.5, 315-331.

1982, Anal. Biochem.

S. Lopez and 0. Mutioz,

and U. Desselberger.

119, 325-329. 1980, Inf. Immunol.

1983, J. Med. Viral.

A.J.. N. Inglis, C.K. Ojeh, D.R. Snodgrass

28, 230-237.

11, 39-52.

and J.D. Menzies,

1982, J. Clin. Microbial.

16,473-

477. Hirt, B. (1967) J. Mol. Biol. 26, 365-369. Jensen,

R.H. and N. Davidson,

Lonsdale,

D.M.,

Maniatis, Spring

T., E.F. Fritsch and J. Sambrook,

1982, Molecular Harbor.

Laboratory,

C.R., R.C. Schwitzer

Merril,

C.R., D. Goldman,

Morrissey,

J.H.,

Cold Spring

and H.L. van Keuren, S.A. Sedman

1981, Anal.

B.R., D.R. Kirsch

Biochem.

and E.E. Nishizawa,

P.M. Slocombe Shatkin,

T. Friedman,

and M. Smith,

L.L. and K. Wang,

H.E., S.E. Straus

Wadell,

Sci. U.S.A.

G. and J.C. de Jong,

p, 104. Cold

76, 4335-4339.

Biochem.

105, 361-363.

17, 876-884. 1981, Electrophoresis

G.N. Air, B.G. Barrell,

2, 135-141.

N.L. Brown,

J.C. Fiddes,

1978, J. Mol. Biol. 125, 225-246. 1981, Biochem.

and C.F. Garon,

Tsai, C.M. and C.E. Frasch,

Manual,

1981, Science 211, 1437-1438.

A.J., J.D. Sipe and P.C. Loh, 1968, J. Viral. 2, 986-991.

Somerville, Takiff,

L.D. Adams

1979, Proc. Natl. Acad.

1980, Anal.

Sammons,

D.W.,

A Laboratory

117, 307-310.

and N.R. Morris, 1976, J. Viral.

F., R. Coulson,

Cloning.

and M.H. Ebert,

Palese, P. and J.L. Schulman, Sanger,

4, 17-32.

I, 849-852.

Harbor

Merril,

Oakley,

1966, Biopolymers

1979, Lancet

1982, Anal.

Biophys.

1981, Lancet Biochem.

1980, Inf. Immunol.

Res. Comm. II, 832-834.

119, 115-l 19. 27, 292-296.

102, 53-58.

C.A. Hutchison,