Rev. sci. tech. Off. int. Epiz., 1996, 15 (3), 875-882
Comparison of a radioactive and
non-radioactive method for sequencing
foot and mouth disease virus isolates
W.C. CARPENTER *, D.V. RAI **, A.R. S A M U E L * and M.C. HÖFNER *
Summary: The authors compare the radioactive method of detecting foot and
mouth disease virus sequence products with a non-radioactive,
silver stain
sequencing
method. The latter was found to compare favourably
to the
radioactive technique for detecting such products. The silver stain
sequencing
method was simple and did not require expensive specialised equipment.
This
new approach will be particularly
useful in developing countries, since the
method does not depend on the availability of fresh radioactive isotopes and
is also safer and considerably
cheaper.
K E Y W O R D S : Aphthovirus - C y c l e sequencing - Foot and m o u t h disease P o l y m e r a s e chain reaction - Silver stain sequencing.
INTRODUCTION
F o o t a n d m o u t h d i s e a s e ( F M D ) is o n e of t h e m o s t e c o n o m i c a l l y i m p o r t a n t v i r u s
diseases of l i v e s t o c k , affecting cattle, s h e e p a n d p i g s . F M D is e n z o o t i c in m o s t a r e a s
of Asia, Africa, t h e M i d d l e E a s t a n d t h e n o r t h e r n p a r t of S o u t h A m e r i c a . A n i m p o r t a n t
r e q u i r e m e n t for t h e c o n t r o l of this d i s e a s e is t h e ability t o i n v e s t i g a t e r a p i d l y t h e likely
source of o u t b r e a k s .
M o l e c u l a r e p i d e m i o l o g y , w h i c h a n a l y s e s t h e g e n o m e of field strains of F M D v i r u s
( F M D V ) b y t h e c o m p a r i s o n of n u c l e o t i d e s e q u e n c e s , is n o w a m a j o r t o o l in t h e s e
investigations. H o w e v e r , s e q u e n c e a n a l y s i s is r e s t r i c t e d to l a b o r a t o r i e s that h a v e e i t h e r
ready a c c e s s to fresh r a d i o i s o t o p e s o r e x p e n s i v e s p e c i a l i s t e q u i p m e n t , s u c h as
automatic s e q u e n c e r s (for n o n - r a d i o a c t i v e s e q u e n c i n g ) . R e c e n t l y , w i t h t h e a d v e n t of
the c y c l e s e q u e n c i n g t e c h n i q u e ( 4 ) , it h a s b e e n p o s s i b l e to m a n u f a c t u r e kits w h i c h are
easy to u s e for s e q u e n c i n g a n d w h i c h c a n e m p l o y either r a d i o i s o t o p e s o r less s e n s i t i v e
m e t h o d s of s e q u e n c e p r o d u c t d e t e c t i o n , s u c h as silver s t a i n i n g .
R a d i o a c t i v i t y is t h e m o s t s e n s i t i v e m e t h o d of d e t e c t i n g s e q u e n c e p r o d u c t s .
However, t h e u s e of this m e t h o d p r e s e n t s difficulties s i n c e s t a t u t o r y r e g u l a t i o n s for t h e
h a n d l i n g of r a d i o a c t i v e i s o t o p e s a r e s t r i n g e n t . M o r e o v e r , t h e half-lives of t h e
radioactive i s o t o p e s n o r m a l l y u s e d for s e q u e n c i n g ( P , S ) a r e fairly s h o r t a n d a r e
therefore u n s u i t a b l e i n c o u n t r i e s w h e r e fresh s u p p l i e s c a n n o t b e g u a r a n t e e d . A n
i m p r o v e d m e t h o d of d e o x y r i b o n u c l e i c acid ( D N A ) s e q u e n c i n g , l i n e a r p o l y m e r a s e
3 2
3 5
* Institute for Animal Health, Pirbright Laboratory, Pirbright, Surrey GU24 ONF, United Kingdom
(A.R. Samuel: corresponding author).
** Indian Veterinary Research Institute, Mukteswar-Kumaon, Nainital, 263138 Uttar Pradesh, India.
876
chain reaction (PCR) cycle sequencing, described by M u r r a y (4), can produce large
a m o u n t s of s e q u e n c e p r o d u c t s , a l l o w i n g less s e n s i t i v e m e t h o d s of d e t e c t i o n t o b e
developed.
I n this p a p e r , t h e a u t h o r s h a v e c o m p a r e d t h e u s e of a n o n - r a d i o a c t i v e silver s t a i n i n g
m e t h o d of v i s u a l i s i n g s e q u e n c e p r o d u c t s w i t h a r a d i o a c t i v e m e t h o d of d e t e c t i o n .
MATERIALS AND METHODS
I n o r d e r t o o b t a i n a t e m p l a t e for s e q u e n c e a n a l y s i s , a 2 1 6 1 b a s e p a i r (bp) f r a g m e n t
f r o m t h e c a p s i d c o d i n g r e g i o n of t h e field i s o l a t e F M D V t y p e О S a u d i A r a b i a 1 5 / 8 8
w a s amplified b y r e v e r s e - t r a n s c r i p t i o n P C R ( R T - P C R ) . T h e m e t h o d w a s as d e s c r i b e d
p r e v i o u s l y (2). P r i m e r p a i r s M H 2 / A R S 3 a n d A R S 4 / N K 6 1 (Table I) amplified
f r a g m e n t s of 1 0 9 6 b p a n d 1301 b p , r e s p e c t i v e l y (Fig. 1). T h e s e p r i m e r p a i r s h a v e b e e n
u s e d successfully to amplify f r a g m e n t s f r o m g e n e t i c a l l y d i v e r s e i s o l a t e s of t y p e О
F M D V . A m p l i f i e d D N A w a s purified f r o m t h e P C R r e a c t a n t s u s i n g W i z a r d T M P C R
purification c o l u m n s ( P r o m e g a , U n i t e d K i n g d o m [ U K ] ) , a c c o r d i n g to t h e i n s t r u c t i o n s
of t h e m a n u f a c t u r e r , o r b y e l e c t r o p h o r e s i s i n l o w m e l t i n g - p o i n t a g a r o s e gels ( 2 ) .
TABLE I
Oligonucleotide
primers
used to evaluate
sequencing
kits
Primer
Orientation
Sequence 5'-3'
NK61 *
Anti-virus sense
GACATCTCCTCCTGCATCTG
2B
NK64 **
Anti-virus sense
GAAGGGCCCAGGGTTGGAGTC
2A
NK30
Anti-virus sense
ATGGCACCGTAGTTGAA
ID
ARS3
Anti-virus sense
CATGTAACGGCGCCTTCGCGTC
1С
ARS4
Virus sense
ACCAACCTCCTTGATGTGGCT
1С
ARS5
Anti-virus sense
CCACAAGGGTCCAAGGC
1B
ARS8
Anti-virus sense
CCGTCGCTACAGGCCACAGGGA
1С
ARS 9
Anti-virus sense
GCGGTTGACGCCAACAAAGGGCAC
1B
ARS 10
Anti-virus sense
TGGTTGGAGGAACTACACCGA
1С
MH2 **
Virus sense
CACACAACCAACACCCAGAACAAT
1A
Location
Most of the primers are anti-virus sense as they were originally designed to be used with viral ribonucleic
acid as a template. Primers NK30 and ARS5 only produced sequences at the lower annealing temperature of
42°C
*
This primer was only used in the polymerase chain reaction to obtain one sequencing template
** These primers equate to PI and P2 respectively from Höfner et al. (2)
Purified p r o d u c t s w e r e s e q u e n c e d u s i n g e i t h e r S i l v e r S e q u e n c e ™ o r f m o l ™
s e q u e n c e kits ( P r o m e g a , U K ) , a c c o r d i n g t o t h e i n s t r u c t i o n s of t h e m a n u f a c t u r e r . B o t h
s e q u e n c i n g kits c o m b i n e d i d e o x y t e r m i n a t i o n s e q u e n c i n g w i t h P C R t e c h n o l o g y to
s e q u e n c e D N A (7). T h i s t e c h n i q u e i n v o l v e s u s i n g o n l y o n e p r i m e r in t h e p r e s e n c e of
d e o x y a n d d i d e o x y n u c l e o s i d e s a n d c y c l i n g t h e r e a c t i o n m a n y t i m e s , t h u s r e - u s i n g the
t e m p l a t e t o p r o d u c e m u l t i p l e c o p i e s of d i d e o x y n u c l e o s i d e - t e r m i n a t e d D N A of v a r y i n g
877
Capsid
5 ' ncr
1A
1С
1B
1D 2 A 2 B n/s
Polymerase
3'ncr
l-AAAAA
VPG-CCCC
Region of genome amplified using overlapping primer sets
MH2
>
ARS4
I
>
1301 bp
2161 bp
ARS3
1096 bp
NK61
FIG. 1
A schematic diagram of the foot and m o u t h disease virus genome,
with the region amplified by polymerase chain reaction and the
location of the primers highlighted
lengths. T h e i n c r e a s e in t h e a m o u n t of D N A p r o d u c e d is n o t l o g a r i t h m i c , as w i t h P C R ,
but linear, as o n l y o n e p r i m e r is i n c l u d e d in e a c h r e a c t i o n . I n o r d e r t o s h o w t h a t t h e
s e q u e n c i n g kits w o r k e d w i t h different t e m p l a t e / p r i m e r c o m b i n a t i o n s , t h e kits w e r e
evaluated u s i n g n i n e o l i g o n u c l e o t i d e p r i m e r s w i t h t h e s i n g l e t e m p l a t e (Table I ) . T h e
nucleotide s e q u e n c e of t h e field i s o l a t e u s e d in this s t u d y ( O / S A U / 1 5 / 8 8 ) h a d n o t
previously b e e n d e t e r m i n e d .
T h e Silver S e q u e n c e ™ kit c o n t a i n e d all t h e r e a g e n t s n e c e s s a r y to p e r f o r m s e q u e n c e
reactions a n d stain t h e g e l s , e x c e p t for t h e specific p r i m e r s w h i c h a r e s u p p l i e d b y t h e
user. T h e f m o l ™ kit s u p p l i e d s e q u e n c e r e a g e n t s a n d D N A p o l y n u c l e o t i d e k i n a s e for
end-labelling p r i m e r s w i t h r a d i o a c t i v e P y - A T P (specific o l i g o n u c l e o t i d e p r i m e r s a r e
supplied b y t h e u s e r ) .
3 2
O w i n g t o t h e fact t h a t t h e f m o l ™ D N A s e q u e n c i n g s y s t e m u s e d r a d i o a c t i v e l y
labelled p r i m e r s t o v i s u a l i s e s e q u e n c e p r o d u c t s , as little as 10 f e m t o m o l e s (fmoles) of
template a n d 4 0 c y c l e s p r o d u c e d sufficient c o p i e s for s e q u e n c e p r o d u c t d e t e c t i o n . T h e
Silver S e q u e n c e ™ kit r e l i e d o n d i r e c t s t a i n i n g of t h e D N A in t h e gel a n d t h u s m o r e
template - 1 p i c o m o l ( p m o l ) - a n d m o r e c y c l e s ( 6 0 ) w e r e r e q u i r e d for s e q u e n c e
product d e t e c t i o n .
W h e n u s i n g t h e f m o l ™ kit s t a n d a r d p r o c e d u r e s w e r e e m p l o y e d for fixing, d r y i n g
and v i s u a l i s i n g t h e r a d i o a c t i v e l y l a b e l l e d s e q u e n c e p r o d u c t s (6). T h e S i l v e r
S e q u e n c e ™ kit r e q u i r e d t h e gel to b e c o v a l e n t l y b o u n d to o n e of t h e s u p p o r t p l a t e s
during c a s t i n g . T h i s i n v o l v e d t h e p r e - t r e a t m e n t of o n e p l a t e w i t h B i n d S i l a n e ™ a n d
the o t h e r w i t h R e p e l c o t e ™ . A f t e r e l e c t r o p h o r e s i s t h e g l a s s p l a t e s w e r e s e p a r a t e d . T h e
gel r e m a i n e d a t t a c h e d to t h e B i n d S i l a n e ™ - t r e a t e d p l a t e , a n d s t a i n i n g p r o c e d u r e s w e r e
878
p e r f o r m e d o n this p l a t e . T h e e x a c t p r o t o c o l is d e s c r i b e d c o m p r e h e n s i v e l y in the
m a n u a l s u p p l i e d w i t h t h e kit, a n d i n c l u d e s a p r o b l e m - s o l v i n g g u i d e . Briefly, t h e b o u n d
gel w a s fixed u s i n g 1 0 % v/v a c e t i c acid in a q u e o u s s o l u t i o n . T h i s gel w a s s t a i n e d w i t h
silver n i t r a t e / f o r m a l d e h y d e s o l u t i o n a n d t h e stain d e v e l o p e d u s i n g a s o d i u m c a r b o n a t e
s o l u t i o n . T h i s c a u s e d t h e silver b o u n d to t h e D N A to p r e c i p i t a t e . D e v e l o p m e n t w a s
s t o p p e d b y further a d d i t i o n of fixative. T h e m a i n r e q u i r e m e n t s for this p r o c e d u r e w e r e
w a t e r a n d r e a g e n t s of h i g h purity. A p p r o x i m a t e l y 15 litres of h i g h - p u r i t y w a t e r ( d o u b l e
distilled) w e r e r e q u i r e d in t h e s t a i n i n g p r o c e s s of e a c h gel.
N i n e oligonucleotide primers c o m p l e m e n t a r y to different sequences within the 2 1 6 1 bp
f r a g m e n t w e r e u s e d for s e q u e n c i n g (Fig. 2 ) . S e v e n of t h e s e p r i m e r s c o u l d b e u s e d w i t h
either kit, e m p l o y i n g t h e s a m e t h e r m a l profile ( 9 4 ° C for 1 m i n u t e , 5 5 ° C for
1 m i n u t e , 7 0 ° C for 1 m i n u t e ) , a l t h o u g h t h e n u m b e r of c y c l e s differed for e a c h m e t h o d
as p r e v i o u s l y m e n t i o n e d . W h e n u s i n g t w o of t h e p r i m e r s ( N K 3 0 a n d A R S 5 ) in the
silver s e q u e n c e kit, t h e a n n e a l i n g t e m p e r a t u r e h a d to b e l o w e r e d f r o m 5 5 ° C t o 4 2 ° C
to o b t a i n a r e a d a b l e s e q u e n c e . T h e s e p r i m e r s w e r e t h e s h o r t e s t p r i m e r s u s e d (Table I),
a n d h a d l o w e r m e l t i n g t e m p e r a t u r e s t h a n t h e o t h e r s , w h i c h m a y h a v e c a u s e d t h e m to
a n n e a l less efficiently at t h e h i g h e r t e m p e r a t u r e . T h e s u b s e q u e n t d e c r e a s e in the
efficiency of t h e c y c l e s e q u e n c e r e a c t i o n r e s u l t e d in less p r o d u c t b e i n g f o r m e d . T h i s
w a s p a r t i c u l a r l y n o t i c e a b l e w h e n u s i n g t h e S i l v e r S e q u e n c e ™ kit, s i n c e m o r e p r o d u c t
is r e q u i r e d for v i s u a l i s a t i o n m e t h o d s i n c o m p a r i s o n w i t h r a d i o a c t i v e m e t h o d s .
1301 bpVirus sense primers
M
H
2
5'LZ
Anti-virus sense primers
ARS9
ARSS
ARSS
ARSIO
ARS3
NK3O NK64
NK6I*
1096 bp
* This primer was not used for sequencing
FIG. 2
The relative position o n the foot and m o u t h disease virus genome
of the oligonucleotides used
RESULTS
U s i n g S i l v e r S e q u e n c i n g ™ , 2 0 0 - 3 0 0 b p c o u l d b e r e a d f r o m a gel ( d e p e n d i n g o n the
p r i m e r u s e d ) . A s i m i l a r n u m b e r of b a s e s w e r e r e a d a b l e u s i n g r a d i o a c t i v i t y . In b o t h
systems, s o m e primers produced better quality sequence data than others, highlighting
t h e i m p o r t a n c e of p r i m e r d e s i g n w h e n c y c l e s e q u e n c i n g . T h e p r i m e r s u s e d in this study
w e r e o r i g i n a l l y d e s i g n e d for direct viral r i b o n u c l e i c acid ( R N A ) s e q u e n c i n g , w h i c h
w a s p e r f o r m e d at 4 2 ° C (3). T h i s fact m a y e x p l a i n w h y s o m e o l i g o n u c l e o t i d e p r i m e r s
w e r e less efficient w h e n u s e d at t h e h i g h e r a n n e a l i n g t e m p e r a t u r e of 5 5 ° C .
R a d i o a c t i v e c y c l e s e q u e n c i n g results w e r e r e c o r d e d as a u t o r a d i o g r a p h s . E x p o s u r e
t i m e i n c r e a s e d t h e l e n g t h of t h e p r o c e s s b y a f e w h o u r s to as m a n y as s e v e r a l d a y s .
879
H o w e v e r , it w a s p o s s i b l e to v a r y e x p o s u r e t i m e s in o r d e r to o b t a i n r e a d a b l e s e q u e n c e s
from r e a c t i o n s t h a t h a d r u n w i t h v a r y i n g d e g r e e s of efficiency o n t h e s a m e gel. R e s u l t s
from silver s e q u e n c i n g gels w e r e s t a i n e d directly, u s i n g s i l v e r n i t r a t e . T h i s w a s l a b o u r
intensive b u t a r e s u l t w a s o b t a i n e d w i t h i n t w o h o u r s of t h e gel b e i n g ran. T h e m a i n
d r a w b a c k s of s i l v e r s t a i n i n g w e r e t h a t l a r g e a m o u n t s ( a p p r o x i m a t e l y 15 litres) of
h i g h - p u r i t y w a t e r w e r e r e q u i r e d for e a c h gel, a n d it w a s n o t p o s s i b l e t o v a r y
d e v e l o p i n g t i m e s b e t w e e n different s a m p l e s o n t h e s a m e gel. T h e i n t e n s i t y of t h e
staining v a r i e d f r o m gel to g e l , d e p e n d i n g o n t h e efficiency of t h e c y c l e s e q u e n c i n g
reaction a n d h e n c e t h e a m o u n t of D N A w h i c h h a d b e e n p r o d u c e d . C o n s i s t e n t s t a i n i n g
was o n l y a c h i e v e d w h e n t h e c y c l e s e q u e n c i n g p r o g r a m m e w a s o p t i m a l l y m a t c h e d to
the p r i m e r s u s e d , a n d w h e n t h e i n s t r u c t i o n s of t h e m a n u f a c t u r e r w e r e f o l l o w e d
carefully. F o r e x a m p l e , if t h e d e v e l o p i n g m i x t u r e w a s n o t c o o l e d t o t h e c o r r e c t
t e m p e r a t u r e ( 1 0 - 1 2 ° C ) , h i g h b a c k g r o u n d s t a i n i n g o c c u r r e d , m a k i n g it difficult to r e a d
the gels. S e q u e n c e d a t a w e r e r e c o r d e d e i t h e r b y air d r y i n g t h e g e l a n d r e a d i n g directly,
or b y r e c o r d i n g t h e gel o n e l e c t r o p h o r e s i s - d u p l i c a t i n g film ( E D F ) . T h e latter w a s
a c h i e v e d b y e x p o s i n g t h e film to fluorescent light (a c o n v e n t i o n a l light b o x ) , u s i n g t h e
stained gel to s h a d o w t h e film. T h e l o n g e r this t y p e of film is e x p o s e d to light, t h e
clearer it b e c o m e s , t h u s s h a d o w s c a s t b y t h e s t a i n e d s e q u e n c e p r o d u c t s r e s u l t e d in d a r k
b a n d s o n t h e E D F . E x p o s u r e t i m e s ( u s u a l l y b e t w e e n 10 to 2 0 s e c o n d s ) w e r e o b t a i n e d
empirically for i n d i v i d u a l g e l s , d e p e n d i n g o n t h e i n t e n s i t y of t h e s t a i n e d s e q u e n c e
p r o d u c t s a n d t h e b a c k g r o u n d s t a i n i n g . H o w e v e r , this m e t h o d w a s f o u n d unsatisfactory,
as it u s u a l l y r e s u l t e d in t h e w a s t e of at least o n e s h e e t of film, g r e a t l y a d d i n g t o t h e
e x p e n s e of t h e t e c h n i q u e . G e n e r a l l y , it w a s e a s i e r to r e a d t h e gels directly. H o w e v e r ,
the m a n u f a c t u r e r states t h a t t h e u s e of E D F c a n e n h a n c e faint b a n d s , t h u s m a k i n g t h e
t e c h n i q u e m o r e s e n s i t i v e . T h e p r e p a r a t i o n of g e l s s t a i n e d to t h e s a m e d e g r e e , a n d t h e
use of a light b o x g i v i n g a n e v e n light d i s t r i b u t i o n , s h o u l d r e s u l t in c o n s i s t e n t
photographic records.
S o m e differences w e r e n o t e d b e t w e e n r a d i o a c t i v e l y l a b e l l e d a n d s i l v e r - s t a i n e d g e l s .
T h e latter c o u l d b e r e a d m o r e e a s i l y at t h e t o p of t h e gel, s i n c e t h e l a r g e r p r o d u c t s ,
w h i c h r u n c l o s e r t o g e t h e r at t h e t o p o f t h e g e l , w e r e m o r e d i s c r e t e . B a n d s o n t h e
a u t o r a d i o g r a p h s w e r e less d i s e r e t e , d u e to s c a t t e r i n g of t h e r a d i o a c t i v e e m i s s i o n s , a n d
b a n d s w h i c h w e r e c l o s e to o n e a n o t h e r m e r g e d . H o w e v e r , w h e n u s i n g silver nitrate,
smaller p r o d u c t s c l o s e r t o t h e p r i m e r d i d n o t stain as w e l l as t h e l a r g e r p r o d u c t s . T h i s
p h e n o m e n o n w a s n o t as p r o n o u n c e d w h e n u s i n g r a d i o i s o t o p e s .
DISCUSSION
It w a s f o u n d t h a t r a d i o a c t i v i t y w a s t h e m o r e r o b u s t of t h e t w o m e t h o d s , s i n c e t h e r e
w a s n o n e c e s s i t y t o v a r y c y c l i n g p r o g r a m m e s to suit different p r i m e r s . M o r e o v e r ,
when using Silver Sequencing™, no readable sequence was obtained w h e n a thermal
cycler w i t h s l o w r a m p t i m e s for h e a t i n g a n d c o o l i n g ( g r e a t e r t h a n o n e d e g r e e C e l s i u s
per s e c o n d ) w a s u s e d . S o m e o l d e r t h e r m a l c y c l e r s a r e t h u s n o t s u i t a b l e for this
t e c h n i q u e . S u c h a d e c r e a s e in efficiency of t h e s e q u e n c e r e a c t i o n , c a u s e d b y t h e s l o w e r
ramp times, was not observed w h e n using the radioactive system.
T h e c o m p a r a t i v e c o s t of S i l v e r S e q u e n c i n g ™ w a s a p p r o x i m a t e l y 1 1 % less p e r
reaction t h a n t h e c o s t of r a d i o a c t i v i t y (8). T h i s d i d not t a k e i n t o a c c o u n t t h e c o s t of
m o n i t o r i n g a n d d i s p o s a l of w a s t e r e a g e n t s , w h i c h is m u c h h i g h e r w i t h r a d i o i s o t o p e s .
880
If s i l v e r - s t a i n e d gels w e r e n o t r e c o r d e d o n E D F , t h e c o s t of e a c h r e a c t i o n w a s a l m o s t
h a l v e d . C o s t is e s p e c i a l l y i m p o r t a n t in d e v e l o p i n g c o u n t r i e s , w h e r e funds m a y b e
l i m i t e d a n d fresh r a d i o i s o t o p e s a r e e x p e n s i v e a n d difficult to o b t a i n . R e a g e n t s u s e d in
silver s e q u e n c i n g h a v e l o n g s t o r a g e l i v e s , w h i c h is n o t t h e c a s e w i t h i s o t o p e s
c o m m o n l y u s e d in s e q u e n c i n g . S i l v e r s t a i n i n g is a l s o safer for l a b o r a t o r y w o r k e r s a n d
d o e s n o t p o s e as g r e a t a h a z a r d to t h e e n v i r o n m e n t w h e n r e a g e n t s a r e d i s p o s e d of. T h i s
safety factor is v e r y i m p o r t a n t , a n d is sufficient r e a s o n t o u s e S i l v e r S e q u e n c i n g ™ .
A l t e r n a t i v e n o n - r a d i o a c t i v e m e t h o d s of d e t e c t i o n a r e a v a i l a b l e for s e q u e n c e
p r o d u c t s . T h e s e i n c l u d e l a b e l l i n g p r i m e r s w i t h fluorescent d y e s w h i c h c a n b e d e t e c t e d
u s i n g e x p e n s i v e s p e c i a l i s e d e q u i p m e n t (5), o r l a b e l l i n g p r i m e r s w i t h h a p t e n s , s u c h as
d i g o x i g e n i n o r biotin, w h i c h c a n b i n d specific h a p t e n a n t i b o d i e s c o n j u g a t e d to
e n z y m e s . T h e latter p r o c e s s i n v o l v e s b l o t t i n g a s e q u e n c e f r o m t h e gel o n t o a
m e m b r a n e , c r o s s - l i n k i n g t h e n u c l e i c a c i d s to t h e m e m b r a n e , i n c u b a t i n g w i t h t h e
a n t i b o d y c o n j u g a t e , a d d i n g s u b s t r a t e s s u c h as L u m i g e n ™ (a c h e m i l u m i n e s c e n t
s u b s t r a t e of a l k a l i n e p h o s p h a t a s e e n z y m e ) a n d e x p o s i n g a film to t h e m e m b r a n e (1).
Silver S e q u e n c i n g ™ has an advantage over the other two non-radioactive methods,
in t h a t it is a q u i c k a n d s i m p l e t e c h n i q u e , a n d d o e s n o t r e q u i r e c a p i t a l o u t l a y or
s p e c i a l i s t e q u i p m e n t w h i c h is n o t a l r e a d y a v a i l a b l e in a m o d e r n m o l e c u l a r b i o l o g y
laboratory.
CONCLUSION
T h e S i l v e r S e q u e n c i n g ™ m e t h o d p r o v i d e d a useful a l t e r n a t i v e t o r a d i o i s o t o p e s for
s e q u e n c i n g F M D viral n u c l e i c acid. T h i s m e t h o d i s , h o w e v e r , n o t as r o b u s t as
r a d i o a c t i v e s e q u e n c i n g . In a d d i t i o n , it r e q u i r e s t h e w o r k e r to b e m o r e a w a r e of t h e
d y n a m i c s of c y c l i n g r e a c t i o n s a n d s u c h factors as t e m p l a t e c o n c e n t r a t i o n a n d p r i m e r
d e s i g n , w h i c h i n f l u e n c e t h e efficiency of t h e s e q u e n c e r e a c t i o n s . W o r k e r s s h o u l d also
b e a w a r e t h a t s o m e i t e m s of e q u i p m e n t , for e x a m p l e t h e r m a l c y c l e r s , w h i c h w o r k w e l l
for r a d i o a c t i v e s e q u e n c i n g m a y n o t b e s u i t a b l e w h e n u s e d for S i l v e r S e q u e n c i n g ™ .
T h e a u t h o r s h a d difficulty o b t a i n i n g g o o d - q u a l i t y h a r d c o p i e s of g e l s , as t h e light b o x
g a v e u n e v e n light d i s t r i b u t i o n w h i c h r e s u l t e d i n i n c o n s i s t e n t E D F e x p o s u r e s . A
h i g h - q u a l i t y light b o x that d i s t r i b u t e s light e v e n l y w o u l d rectify this p r o b l e m . A s silver
s e q u e n c i n g is c h e a p e r , d o e s n o t rely o n t h e availability of fresh r a d i o i s o t o p e s a n d is
safer to u s e , this t e c h n i q u e w o u l d b e e s p e c i a l l y v a l u a b l e for t h e transfer of s e q u e n c i n g
t e c h n o l o g y to d e v e l o p i n g c o u n t r i e s .
ACKNOWLEDGEMENTS
T h e a u t h o r s t h a n k M r N . J . K n o w l e s for d e s i g n i n g a n d s u p p l y i n g s o m e of t h e
p r i m e r s . T h a n k s a r e also d u e to D r J.R. C r o w t h e r for his h e l p in t h e p r e p a r a t i o n of t h e
m a n u s c r i p t . T h i s w o r k w a s partially s u p p o r t e d b y t h e M i n i s t r y of A g r i c u l t u r e ,
Fisheries and Food, United Kingdom.
*
* *
881
COMPARAISON
ENTRE
LES MÉTHODES
RADIOACTIVE
ET NON
RADIOACTIVE P O U R L E S É Q U E N Ç A G E D E S ISOLATS D U VIRUS D E L A
FIÈVRE A P H T E U S E . - W.C. Carpenter, D.V. Rai, A.R. Samuel et M . C . Höfner.
Résumé : Les auteurs comparent la méthode radioactive d'identification
des
produits de séquençage
du virus de la fièvre aphteuse à une méthode de
séquençage non radioactive par imprégnation à l'argent. Ils en concluent que
cette dernière offre le plus d'avantages pour la détection de tels produits. Elle
est, en effet, simple et ne nécessite pas d'instrumentation
coûteuse et
sophistiquée. De plus, cette nouvelle méthode sera particulièrement
utile dans
les pays en développement
car elle ne nécessite pas d'isotopes
radioactifs
frais ; elle est également moins dangereuse et bien meilleur
marché.
M O T S - C L É S : Amplification e n chaîne p a r p o l y m é r a s e - Aphtovirus Fièvre aphteuse - S é q u e n ç a g e p a r cycle - S é q u e n ç a g e p a r imprégnation à
l'argent.
*
COMPARACIÓN D E UN MÉTODO RADIOACTIVO Y OTRO N O RADIOACTIVO
PARA L A S E C U E N C I A C I Ó N D E A I S L A D O S D E L V I R U S D E L A F I E B R E A F T O S A .
- W.C. Carpenter, D.V. Rai, A.R. Samuel y M . C . Höfner.
Resumen: Los autores comparan el método radioactivo para la detección de
productos
secuenciales
del virus de la fiebre aftosa con un método de
secuenciación
no radioactivo,
basado en una tinción de plata. Este último
resultó más adecuado para la detección de dicho tipo de productos. El método
de secuenciación
por tinción de plata es sencillo y no exige disponer de
material muy caro y especializado.
Este nuevo enfoque puede
resultar
especialmente
útil en los países en vías de desarrollo, pues su aplicación no
depende de la disponibilidad
de isótopos radioactivos frescos, además de
resultar más seguro y considerablemente
más barato.
P A L A B R A S C L A V E : Aftovirus - Fiebre aftosa - R e a c c i ó n d e
polimerización e n c a d e n a - Secuenciación cíclica - Secuenciación p o r
tinción d e plata.
*
* *
REFERENCES
1.
C R E A S Y A., D ' A N G I O L. J R , D U N N E T . S . , K I S S I N G E R С , O ' K E E F F E T . , P E R R Y - O ' K E E F F E
H . , M O R A N L . S . , ROSKEY M . , SCHILDKRAUT I., SEARS L . E . & SLATKO B . ( 1 9 9 1 ) .
-
Application of a novel chemiluminescent-based D N A detection method to single-vector
and multiplex D N A sequencing. Biotechniques,
11 ( 1 ) , 1 0 2 - 1 0 9 .
2.
H Ö F N E R M . C . , C A R P E N T E R W . C . & D O N A L D S O N A.I. ( 1 9 9 3 ) . -
D e t e c t i o n of
foot-and-
mouth disease virus R N A in clinical samples and cell culture isolates by amplification of
the capsid coding region. J. virol. Meth., 42, 5 3 - 6 2 .
882
3. K N O W L E S N . J . (1990). - A method for direct nucleotide sequencing of foot-and-mouth
disease virus R N A for epidemiological studies. In Report of the Session of the Research
Group of the Standing Committee of the European Commission for the Control of
Foot-and-mouth Disease. Lindholm, Denmark, 25-29 June. Food and Agriculture
Organisation (FAO), R o m e , Appendix 06-112.
4. M U R R A Y V. (1989). - Improved double-stranded D N A sequencing using the linear
polymerase chain reaction. Nucleic Acids Res., 17, 8889.
5. NiSHiKWANA T . & K A M B A R A H. (1992). - High resolution-separation of D N A bands by
electrophoresis with long gels in a fluorescent detection D N A sequencer.
Electrophoresis,
13, 495-499.
6. S A M B R O O K J., F R I T S C H E.F. & M A N I A T I S T . (1989). - M o l e c u l a r cloning: a laboratory
manual. Cold Spring Harbor Laboratory Press, N e w York, 645.
7.
SANGER
F., N I C K L E N
S. &
C O U L S O N A.R.
(1977). -
DNA
sequencing
with
chain
termination inhibitors. Proc. Natl Acad. Sci. USA, 7 4 , 5463-5467.
8. W A D E - E V A N S A . (1994). - Plasmid D N A sequencing using a silver staining protocol. In
Laboratory products technology. Promega U K , 9.