ligase chain reaction
TRANSCRIPT
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See discussions, stats, and author profiles for this publication at: http://www.researchgate.net/publication/15024770
Ligase Chain-Reaction (Lcr) – Overview andApplications
ARTICLE in PCR METHODS AND APPLICATIONS · MARCH 1994
DOI: 10.1101/gr.3.4.S51 · Source: PubMed
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1994 3: S51-S64Genome Res. M Wiedmann, W J Wilson, J Czajka, et al.
Ligase chain reaction (LCR)--overview and applications.
References http://genome.cshlp.org/content/3/4/S51.refs.html
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l l | l l l l a n u a l S u p p l em e n
L igase Ch a in
R e a c t i o n
L C R ) - -O v e r v i e w
a n d
A p p l i c a t i o n s
M a r t in W i e d m a n n 1
W e n d y I . W i l s o n 2 J o h n
C z a j k a 1 J ia n y i n g L u o 3
F r a n c i s B a r a n y ~ a n d C a r l
A . B a t t 1
1Department of Food Science,
Cornell University, Ithaca, New
York 14853; 2Department of Plant
Pathology, New York State
Agricultural Experiment Station,
Corneli University, Geneva, New
York 14456; 3Department of
Microbiology, Hearst Microbiology
Research Center, Cornell University
Medical College, New York, New
York 10021
PCR has facilitated the development of a variety of nucleic acid-based detec-
tion systems for genetic disorders as well as for bacterial, viral, and other
pathog ens. (1) In the last few years, a number of oth er DNA amplification
methods, incl udin g self-sustained sequence replicat ion (3SR), (2) Q-beta rep-
licase (QI3), 3) and the ligase chain react ion (LCR), 4 s) have b een developed to
complem ent, or as alternatives to, PCR. (6 7) From its initial detailed reports in
1991, LCR evolved as a very promising diagnostic technique that is often
utilized in conjunction with a primary PCR amplification. LCR employs a
thermostable ligase and allows the discrimination of DNA sequences differing
in only a single base pair (see Fig. 1). (4 s) The power of LCR is its comp atibil ity
with other replication-based amplification metho ds. By comb inin g LCR with
a primary amplification, one effectively lines up the crosshairs to distinguish
single base-pair changes with pinpoint accuracy.
The intellectual genesis of LCR can be traced back to pioneering work by
Whiteley et al. (8~ who described an oligonucleotide probe-based assay using
two probes that are ligated together only whe n immediate ly adjacent to each
other. The same concept is applied in the oligonucleotide ligation assay
(OLA). (9 10)This method was used in conjunction with a primary PCR step to
screen for sickle cell anemia, the AF508 mutation in cystic fibrosis, and T-cell-
receptor poly morp hisms. Wu and Wallace ~11t described a similar techn ique
called the ligase amplificati on reaction (LAR), whic h employs two sets of
complem entary primers and repeated cycles of denatu ration (at 100~ and
ligation (at 30~ using the mesophi lic T4 DNA ligase. Use of mesoph ilic, t hat
is, T4 or Escher i ch ia co l i ligase has the drawback of requiring the addition of
fresh ligase after each denaturation step, as well as appearance of target-
ind epe nde nt ligation products. (11,~2) In contrast, LCR provides a much higher
sensitivity and is less susceptible to the formation of false-positive ligation
products.
Thermostable ligase minimizes target-independent ligation because the
reaction can be performed at or near the melting temperature (T,n) of the
oligonuc leotides. (s) Furthermore , the use of thermos table ligase avoids the
need to add fresh ligase after each denaturation step as required in LAR.
Recently, thermostable ligase has become available from a variety of com-
mercial suppliers, and this will probably lead to even wider application and
use of this new amplification technique.
The concep t of LCR and ligation-based diagnostics has been reviewed. (s 13)
We will provide an overview of the recent advancements, new develo pments,
and applications of LCR and similar ligase-mediated detection methods.
T H E O R Y O F L CR N D S I M I L R M P L I F I C T I O N M E T H O D S
The principle of LCR is based in part on the ligation of two adjacent synthetic
oligonucleotide primers, which uniqu ely hybridize to one strand of the target
DNA (see Fig. 1). The junct ion of the two primers is usually pos ition ed so that
the nucleotide at the 3 end of the upstream p rimer coincides with a potential
single base-pair difference in the targeted sequence. This single base-pair
difference may define two different alleles, species, or other polymorphisms
correlated to a given phenotyp e. If the target nucleotide at that site comple-
ments the nucleotide at the 3 end of the upstream primer, the two adjoining
primers can be covalently joined by the ligase. The unique feature of LCR is
a second pair of primers, almost entirely complementary to the first pair, that
are designed with the nucleotide at the 3 end of the upstream primer denot-
ing the sequence difference. In a cycling reaction, using a thermostable DNA
ligase, both ligated products can then serve as templates for the next reaction
cycle, leading to an exponential amplification process analogous to PCR am-
plification. If there is a mismatch at the primer junction, it will be discrirni-
3:SS1-S649 by Co ld Sprin g Harbor Laboratory ISSN 1054-9805/94 $1.00
I~ ,R
M e t h o d s a n d p p l ic a t io n s
1
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Man u a l Sup p l em en t ll lU lmW
FIGURE 1 Principle of LCR. Bottom) The example shown is an LCR with matched target (L.
monocytogenes) and mismatched target L. innocua). The pathogenic bacter ia L. monocytogenes can
be distinguished from other closely related Listeria spp. (e.g., L. innocua) by a single base-pair
difference in the 16S rDNA/2~ L. monocytogenes has an A-T base pair at nucleotid e 1258, whereas
L. innocua has a G-C base pair at this position. Top) DNA is denat ured at 94~ and the four LCR
primers anneal to their complementa ry strands at 65~ which is approximately 5~ below their
Tin. Therm ostable ligase (Q) will only ligate primers th at are perfectly compl em enta ry to thei r
target sequence and hybridize directly adjacent to each other (as shown with
L. monocytogenes,
left). The discrim inating bases at the 3 ends of the upstream primers are depicted as boxes on the
target as well as on the primers for clarity. Primers that h ave at least a single base-pair mism atch
at the 3 end con tributi ng to the jun ction of the two primers will not ligate (as show n with L.
innocua, right). The discr iminating primers have a 2-bp nonco mple men tary AA tail at their 5
ends to avoid ligation of the 3 ends.
n a t e d a g a i n s t b y t h e r m o s t a b l e l i ga s e a n d t h e p r i m e r s w i l l n o t b e l i g a te d . T h e
a b s e n c e o f t h e l i g a t e d p r o d u c t t h e r e f o r e i n d i c a t e s a t l e a st a s i n g l e b a s e - p a i r
c h a n g e i n t h e t a r g e t s e q u e n c e . ~4) L i g a se d e t e c t i o n r e a c t i o n ( L D R ) i s s i m i l a r t o
L C R . ~s) I n L D R , o n e p a i r o f a d j a c e n t p r i m e r s t h a t h y b r i d i z e t o o n l y o n e o f t h e
t a r g e t s t r a n d s i s u s e d t o a c h i e v e a l i n e a r a m p l i f i c a t i o n ( s ee F ig . 2 ). L D R m a y
b e u s e d f o l l o w i n g a p r i m a r y a m p l i f i c a t i o n ( P CR , 3 SR , Q l 3 - r ep l i c as e , R T - P C R )
a n d h a s t h e a d v a n t a g e o f a c c u r a t e l y q u a n t i t a t i n g t h e r a t i o o f t w o a l l e le s i n a
t a r g e t s a m p l e . ~14) L D R c o u p l e d t o P C R h a s p r o m i s e i n a m u l t i p l e x f o r m a t
w h e r e s e v e r a l m u t a t i o n s a r e a n a l y z e d i n a s i n g l e a m p l i f i c a t i o n . ~s) T h i s
52
PCR Me t h o d s a n d p p l i c a t i o n s
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l l |l l i l an u a i Supp lem en
FIGURE 2 Princ iple of PCR-coupled LDR. The sa me two target sequ ence s as in Fig. 1 are used to
illustrate the PCR-coupled LDR. The DNA stretch co ntain ing the single base-pair difference that
dist inguishes L. monocytogenes from L. innocua (see Fig. 1, bottom is PCR amplified using PCR
primers outsid e the region o f the LCR primers. The PCR amp lifies both target sequen ces und er
standard con dit ions (detai ls can be found in Refs. 20 and 32). ( 9 Taq polymerase 3' to each of
the two PCR primers. After PCR amplification,
Taq
polymerase is inactivated by 97~ for 25
rain. 133~. An aliquo t o f the PCR-amplified DNA (between 1% and 4% o f the PCR reaction) is then
used in the LDR. DNA is denatu red at 94~ and the two LDR prime rs annea l to their comple-
men tary strand at 65~ whic h is appro xima tely 5~ below their Tin. As in LCR (Fig. 1), the
thermostable l igase (Q) wil l only l igate primers that are perfectly comp lemen tary to their target
sequence and hybridize directly adjacent to each other (as shown with L. monocytogenes, left .
Primers that have at least a single base-pair mism atch at the 3' en d co ntributing to the junction
of the two prim ers will not ligate (as show n with L. innocua, right . An LDR cycle, w hich consists
of a den aturin g step at 94~ for 1 rain and an anne alin g step of 65~ is repeated 5-2 0 times so
that a l inear am plification of l igated LDR primers is achieved with the com plem entary target ( i.e.,
L. monocytogenes .
m e t h o d is c u r r e n t l y b e i n g a p p l ie d t o t h e s i m u l t a n e o u s d e t e c t i o n o f m u l t i p l e
m u t a t i o n s i n c y s t i c f i b r o s i s ~15'1 6) a s w e l l a s i n 2 1 - h y d r o x y l a s e d e f i c i e n c y . ~17)
p L C R is a n o t h e r l i g a s e - m e d i a t e d d e t e c t i o n m e t h o d , w h e r e t h e 3 ' e n d s o f
t h e d i s c r i m i n a t i n g ( o r a l l e le - s p e c if i c ) p r i m e r s c o i n c i d e w i t h a p o t e n t i a l b a s e -
p a i r c h a n g e . T h e p L C R p r i m e r s a r e d e s i g n e d w i t h a g a p b e t w e e n t h e d i s c r i m -
i n a t i n g a n d t h e n o n d i s c r i m i n a t i n g p r i m e r ( s ee Fi g. 3 ). In th i s r e a c ti o n , t h e
g a p i s f il l e d u s i n g t h e T a q p o l y m e r a s e S t o f fe l f r a g m e n t , f o l l o w e d b y t h e l i ga -
t i o n o f t h e e l o n g a t e d d i s c r i m i n a t i n g p r i m e r w i t h t h e n o n d i s c r i m i n a t i n g
p r i m e r . T h e s p e c i f i c i t y o f t h i s m e t h o d r e li e s o n a l l e l e -s p e c i f i c e l o n g a t i o n o f
t h e d i s c r i m i n a t i n g p r i m e r b y t h e p o l y m e r a s e . B i r k e n m e y e r a n d M u s h a h -
w a r ~18~ d e s c r i b e d a n o t h e r l i g a s e - m e d i a t e d t e c h n i q u e c a l l e d g a p p e d L C R (G -
L CR ). T h i s t e c h n i q u e u s e s f o u r 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 t w o p r i m e r s ,
o f e a c h p a i r b e i n g s e p a r a t e d b y a g a p o f o n e o r m o r e c o n s e c u t i v e b a s e s t h a t
a r e s p e c i f i c f o r t h e t a r g e t D N A ( s e e Fi g. 4 ). B y a d d i n g o n l y t h e m i s s i n g d e o x -
y n u c l e o t i d e s t o t h e r e a ct i o n t o g e t h e r w i t h a t h e r m o s t a b l e p o l y m e r a s e a n d a
P R Me t h o d s a n d A p p l i c a t i o n s 5 3
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Man ua l Supp lem en t lU
FIGURE 3 Prin ciple of pLCR. The same target sequ enc es as in Fig. 1 are used to illu strate pLCR.
After the d enat uring o f the DNA at 94~ the four pLCR prime rs are allowed to ann eal at 65~
These primers anneal so that a two- or three-nucleotide gap between the primers of one pair
(which anneals to the same strand) is formed. The 3 end o f the d iscriminating primers (shown
with a shaded box at the 3 end and w ith the discriminating nucleotides indicated by T and A)
can be elongated by the Taq polymerase Stoffel fragment and the appropriate nucleotides anal-
ogous to the process in PCR. Onl y the nucleo tides neede d to fill the two- or three-nu cleotid e gap
between the d iscriminating and the no ndiscrim inating primers are included in the reaction mix;
dATP, dGTP, and dTTP are needed for the exam ple shown. After elongation of the discrimin ating
primers by two or three nucleotides, the junction b etween the elonga ted discrim inating primer
and the nond iscrimin ating primer can be sealed by the thermostable l igase. This cycle is repeated
between 30 and 60 t imes. With a noncomplementary target right), no elongation of the dis-
crimina ting primer is possible; therefore, no l igation of the two primers w il l occur and no pLCR
product wil l form.
t h e r m o s t a b l e l ig a s e, t h e g a p m u s t f ir st b e f i l le d i n th e p r e s e n c e o f t h e m a t c h -
i n g t a rg e t a n d b e f o r e t h e r e s u l t i n g n i c k c a n t h e n b e s e a l e d b y t h e l i g a s e. T h i s
t e c h n i q u e l i m i t s i ts e l f t o t h e d e t e c t i o n o f b a s e -p a i r c h a n g e s f r o m A -T FF -A t o
G - C / C - G o r v i c e v e r s a . Fo r e x a m p l e , g a p p e d L C R c o u l d n o t d i s t i n g u i s h 13A
g l o b i n f r o m [ 3 B g l o b i n ( A- -~ T t r a n s v e r s i o n ) b e c a u s e t h e r e i s n o d i f f e r e n c e i n
t h e b a s e s r e q u i r e d f o r f il l i n g t h e g a p . A s i m i l a r p r i n c i p l e i s al s o a p p l i e d i n t h e
r e p a i r c h a i n r e a c t i o n ( RC R ), w h i c h h a s b e e n u s e d f o r t h e d e t e c t i o n o f h u m a n
p a p i l l o m a v i r u s ( H P V) 1 6. (19)
COMPARISON OF LCR pLCR AND G-LCR
O n e p e r f o r m a n c e - l i n k e d d i f f e r e n c e c i te d a m o n g L CR , p L C R, a n d G - L C R i s t h e
r e l a ti v e a m o u n t o f l i g a te d p r o d u c t i n t h e a b s e n c e o f t e m p l a t e . B e c a u s e b o t h
p L C R a n d G -L CR r eq u i r e a n i n i t i a l t e m p l a t e - d e p e n d e n t e x t e n s i o n , t h e y h a v e
b e e n p r o p o s e d t o b e l e ss p r o n e t o f a l se p o s i t i v e s i n t h e a b s e n c e o f t e m p l a t e .
T o c o m p a r e L CR , p L CR , a n d G - LC R , t h e a p p r o p r i a t e p r i m e r s f o r t h e d e t e c t i o n
o f Listeria monocytogenes b y th e s e t h r ee t e c h n i q u e s w e r e d e s i g n e d a n d s y n -
t h e s i z e d . (2~ L o c a t i o n s o f t h e p r i m e r s a r e s h o w n i n F i g u r e s 1 , 3 , a n d 4 .
5 4 PCR Me t h o d s a n d App l i c a t i o n s
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i l IIIIM an u al Supplem en
FIGURE 4 Principle of G-LCR. The same ta rget seq uence s as in Fig. 1 are used to illust rate G-LCR.
After denat uring o f the DNA at 94~ the four primers are allowed to annea l at 65~ These
primers anneal so that a one-nucleotide gap between the pr imers of one pair (which anneals to
the same strand) is formed. This gap is located so that it coincides with the base pair discrimi-
nating the two targets
L. monocytogenes
and
L. innocua
in the example shown) from each other .
The 3 ' end of the dow nstream prim er can be elongated by the
Taq
polymerase Stoffel fragment
and the appropriate nucleotides analogous to the process in PCR. Only the nucleotides needed
to fill the one-nucleotide gap (shown as a shaded box in the target sequence with the discrim-
inating nucleotides indicated by T and A) between the two primers are included in the reaction
mix; only dATP and dTTP are needed for the exam ple show n. After elonga tion of the discrimi-
nating primers with the appropriate nucleotide, the iunction between th e elongated down stream
primer and the upstream primer can be sealed by the thermostable ligase. This cycle is repeated
between 30 and 60 times. With a noncomplementary target right), no elongation of the dis-
crim inatin g prim er is possible; th erefore, no ligation of the two prim ers will occur and no G-LCR
product will form.
R a d i o a c t i v e l y l a b e l e d L CR p r i m e r s a n d d e t e c t i o n o f t h e l i g a t i o n p r o d u c t s a f t e r
g e l e l e c t r o p h o r e s i s ( fo r d e t ai l s, s ee D e t e c t i o n M e t h o d s o f L C R p r o d u c t s )
w e r e u s e d t o c o m p a r e t h e t h r e e m e t h o d s f o r t h e i r a b i l i ty t o d et e c t s i n g l e
b a s e - p a i r d if f e r e n c e s i n P C R - a m p l i f i e d 1 6S rD N A . T h e r e a c t i o n c o n d i t i o n s f o r
G - L C R as w e l l f o r p L C R a r e as d e s c r i b e d i n T a b l e 1 f o r pL C R . I n c o n t r a s t t o
p r e v i o u s r e p o r t s f o r G -L C R , t h e Taq p o l y m e r a s e S t o ff e l f r a g m e n t w a s u s e d
i n s t e a d o f Taq p o l y m e r a s e . C o m p a r e d w i t h Taq p o l y m e r a s e , t h e S t o ff e l f r ag -
m e n t l a c k s 5 ' ~ 3 ' e x o n u c l e a s e a c t i v i t y a n d d o e s n o t e x c i s e b a s e s f r o m t h e 5 '
e n d o f t h e p r i m e r a d j a c e n t t o t h e g a p . N o t a r g e t - i n d e p e n d e n t l i g a t i o n p r o d -
u c t s w e r e o b s e r v e d f o r L C R, p L C R , o r G - L CR . F u r t h e r m o r e , a c l e a r d i f f e r e n t i -
a t i o n o f
L. monocytogenes
f r o m
L. innocua
b a s e d o n a s i n g l e b a s e - p a i r d i f f e r -
e n c e i n t h e 1 6S r D N A w a s p o s s i b l e f o r al l t h r e e f o r m a t s . T h i s i s t h e f i rs t t i m e
t h a t a s i n g l e b a s e - p a i r d i f f e r e n c e w a s d e t e c t e d u s i n g G - L C R . P r e v i o u s r e p o r t s
d e s c r i b e d t h e d i s c r i m i n a t i o n o f t a r g e ts w i t h a t le a st t w o b a s e - p a i r d i f fe r -
e n c e s . ~22'23~ F u r t h e r m o r e , t h i s s h o w s t h a t L C R , p L C R , a n d G - L C R h a v e t h e
p o t e n t i a l t o d e t ec t s i n g l e b a s e- p a i r d i ff e r e n c es ; t h e i r d i s c r i m i n a t o r y a b i l i t y
PCR Me t h o d s a n d A p p l i c a t i o n s 5 5
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M a n u a l S u p p le m e n t l i l I l i
TABLE 1 P r o t o c o l s f o r LC R , p L C R , a n d G - L C R
LCR pLCR G-LCR
De taile d refe renc es 4, 5, 20, 27, 30, 32, 21
41, 42
Po s i t io n o f 3 ' b a se o f b o th 3 ' b a se o f b o th
d i sc r im in a t in g s t r an d s ( s in g le s t r an d s (1 o r 2
n u c leo t id e b a se 3 ' o v e rh an g ) b a se s o v e rh an g )
Tm of pr im ers 66--70~ (4'2~ or 68-70~ 62-76 ~
60-66oc. (42)
Am o u n t o f e ach 1 -1 0 fmo le s /~ l 2 fmo le s /~ l
p r i m e r
Lab e l in g o f p r imers
R e a c t io n v o l u m e
Bu f fe r co n d i t io n s
A m o u n t o f
n u c leo t id e s fo r
f i l l - in r eac t io n
Carrier DNA to
su p p re s s
b a c k g r o u n d
T h e r m o s t a b l e
e n z y m e s / r e a c t i o n
v o l u m e
C y c l e c o n d i t i o n s
b io t in /d ig o x ig en in ; 3 2 p
fluorescein; 32p
10- 50 ~1 25 ~1
20--50 mM Tris-HC1 80 mM KO H/K CI, 50
(pH 7.6), 100 mM mM EPPS, 10 mM
KC1, 10 mM MgCl2, 10 mM
MgCI2, 1 mM NH4C1, 1 mM
EDTA, 10 mM DTT, 10 ~g/m l
DTT, 1 mM NA D*, BSA, 1 mM NAD *
0 .1 -0 .0 1 Tr i to n
X-IO0~
1 ~M
0 .4 ~ g sa lmo n
sperm DNA/p.I
1 .5 n ick c losing
u n i t s
aq
ligase /~l (4'2~ or
0.15 U/p.1 24)
94~ for 1 min , 65~
for 4 ra in , 10-30
cyclesb or 94~ for
1 rain , 60~ for 8
ra in ; 30 cy cles ~
1.5 n ick c losing
u n i t s aq ligase/p.1
an d 0 .0 8 u n i t s
aq
p o ly merase S to f fe l
fra gm en t/~ l (21~
97~ for 3 ra in , 1
cycle; 94~ for 1
min , 65~ for 4
min ; 5 0 cy c le s
22, 23
n u c leo t id e s to b e f i l l ed in
1 6 .6 -2 0 fmo le s /~ l
b io t in / f lu o re sce in ;
u n l a b e l e d , u s e d w i t h
3 ap - lab e led n u c leo t id e s
fo r f i l l - in r eac t io n
2 5 -5 0 ~ l
80 mM KOH/KCI, 50 mM
EPPS, 10 mM M gCl 2,
10 mM NH4CI, 1 mM
DTT, 10 ~xgtml BSA,
0.1 mM NA D §
1 p.M
68 U/~I aq l ig a se an d
0.02 U/~I aq
pol ym era se (22,z3)
h e a t i n b o i l i n g w a t e r
b a th fo r 3 r a in ; th en
85~ for 30 sec,
5 0 -6 0 ~ fo r 2 0 sec -1
ra in ; 2 7 -6 0 cy c le s
aBarany(4) d id not include Tri ton X-100.
bFor primers with Tm of 66-72~176
CFor p r imers wi th Tm of 60 -6 6~ ~4z~
m i g h t n e v e r t h e l e s s d e p e n d o n t h e n a t u r e a n d c o m p o s i t i o n o f t h e t a r g e t s . T h e
s e n s i t i v i t y o f t h e s e t h r e e t e c h n i q u e s i n a c o m p a r a t i v e s t u d y i s c u r r e n t l y u n d e r
i n v e s t i g a t i o n i n o u r l a b o r a t o r i e s .
L CR R E A C T I O N S I M P O R T A N T F AC TO R S
A c c u r a t e r e s u l t s f r o m L C R a s s ay s d e p e n d o n a v a r i e t y o f f a c to r s , i n c l u d i n g
p r i m e r d e s i g n a n d r e a c t i o n c o n d i t i o n s . B a s e d o n o u r e x p e r i e n c e a n d t h o s e o f
o t h e r s o v e r t h e p a s t 3 y e a r s , a f e w of t h e m o s t i m p o r t a n t f a c t o rs t h a t n e e d t o
b e c o n s i d e r e d i n t h e d e v e l o p m e n t o f L C R a s s a y s f o l l o w .
D e s i g n o f LC R P r i m e r s
T o m i n i m i z e t a r g e t - i n d e p e n d e n t l i g a t i o n , L C R p r i m e r s w i t h a s i n g l e b a s e - p a i r
o v e r h a n g , r a t h e r t h a n b l u n t e n d s , s h o u l d b e u s e d . T h e i m p o r t a n c e o f s i n g l e
b a s e - p a i r o v e r h a n g s i s s h o w n b y K / i li n e t a l ., (24) w h o r e p o r t e d a r e l a t i v e l y h i g h
a m o u n t o f t a r g e t - i n d e p e n d e n t l i g a t i o n u s i n g p r i m e r s w i t h b l u n t e n d s . T h e T m
6 P CR M e t h o d s an d A p p l i c a t io n s
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l l l l l l l a n u a l S u p p le m e n
o f al l fo u r p r i m e r s o f o n e s e t of LC R p r i m e r s s h o u l d b e w i t h i n a n a r r o w
t e m p e r a t u r e r a n g e , id e a l ly w i t h a n a b s o l u t e m o f 7 0 ~ -+ 2 ~ F u r t h e r m o r e ,
t h e p r i m e r s s h o u l d b e d e s i g n e d s o t h a t o n e p r i m e r c a n n o t s e r v e a s a b r i d g i n g
t e m p l a t e f o r o t h e r p r i m e r s a n d t h e r e f o r e l e a d t o t a r g e t - i n d e p e n d e n t l i g a ti o n .
A d d i n g n o n c o m p l e m e n t a r y t ai ls o f t w o n u c l e o t id e s o r l o n g e r to t h e n o n a d -
j a c e n t 5 ' e n d s o f t h e p r i m e r s s h o u l d p r e v e n t l i g a t i o n o f t h e 3 ' e n d s . D e p e n d -
i n g o n t h e d i s c r i m i n a t e d n u c l e o t i d e s , d i f f er e n t a m o u n t s o f l i g a t i o n p r o d u c t
a r e o b s e r v e d w i t h a m i s m a t c h e d t a r g e t. (4~ E x p e c t e d a m o u n t s o f f al s e l i g a t i o n
f o r s p e c i f i c m i s m a t c h e s a r e s h o w n i n T a b l e 2 . T h e s e d a t a c a n b e u s e d f o r
d e s i g n i n g p r i m e r s w i t h t h e l o w e s t p o s si b l e r a te o f f a ls e l i g a t i o n w h e n s o m e
c h o i c e b e t w e e n d i f f e r e n t t a r g e t s e q u e n c e s e x i s t s .
T h e n a t u r e o f t h e b a s e p a i r a t t h e 3 ' e n d o f t h e p r i m e r w i t h t h e m a t c h e d
t a r g e t s e e m s t o i n f l u e n c e t h e l i g a t i o n e f f i c i e n c y . T w o s e t s o f L C R p r i m e r s w i t h
t h e c o r r e s p o n d i n g d i f f e r e n c e at t h e 3 ' e n d o f t h e d i s c r i m i n a t i n g p r i m e r w e r e
u s e d f o r t h e d e t e c t i o n o f a s i n g l e b a s e - p a i r d i f f e r e n c e ( D 1 2 8 G ) i n t h e t w o
a l l el e s o f t h e b o v i n e C D 1 8 g e n e ) 2s~ T h e d i s c r i m i n a t i n g p r i m e r s e t t h a t c a r r i e s
a G o n o n e a n d a C o n t h e o t h e r 3 ' e n d g a v e a m o r e e f f i c i e n t l i g a t i o n a s
c o m p a r e d w i t h t h e s e c o n d s et o f p r i m e r s i n w h i c h t h e d i s c r i m i n a t i n g p r i m e r s
c a r ry a n A a n d a T a t th e i r 3 ' e n d s . T h e g r e a t e r h y d r o g e n b o n d i n g o f t h e G - C
b a s e - p a i r i n g f a c i l i ta t e s a m o r e s t a b l e h y b r i d a s c o m p a r e d w i t h A - T b a s e - p a i r -
i n g , t h e r e f o r e a l l o w i n g a m o r e e f f i c i e n t l i g a t i o n .
L R o n d i t i o n s
S t a n d a r d c o n d i t i o n s f o r a 5 0 -t ~1 L C R a r e a s f o l lo w s : O n e s e t o f f o u r p r i m e r s
( b e t w e e n 2 5 a n d 2 0 0 f m o l e s o f e a c h p r i m e r ) is i n c u b a t e d i n th e p r e s e n c e o f
t a r g e t D N A i n t h e r e a c t i o n b u f f e r ( 5 0 m M T r i s- H C 1 a t p H 7 . 6, 1 0 0 m M K C 1 , 1 0
m M M g C1 2, 1 m M ED T A, 1 0 m M d i t h i o t h r e i t o l , 1 m M N A D + , 2 0 ~ g o f s a l m o n
s p e r m D N A ) w i t h 7 5 n i c k - c l o s i n g u n i t s o f hermus aquaticus DN A l igase . (26)
T h e i n c l u s i o n o f 0 . 0 1 - 0 . 1 T r i t o n X -1 0 0 i n t h e r e a c t i o n b u f f er g i v es a
h i g h e r l i g a t i o n r a t e b u t a l s o l e a d s t o a s l i g h t i n c r e a s e o f l i g a t i o n w i t h a m i s -
m a t c h e d t a r g e t. (2 ~ R e a c t i o n c y c le s a r e u s u a l l y 1 5 s e c t o 1 r a i n a t 9 4 ~ f o r
d e n a t u r a t i o n , f o l l o w e d b y 4 m i n t o 6 r a i n at 6 0 - 6 5 ~ ( i d ea l ly 5~ b e l o w t h e
l o w e s t T m o f t h e p r i m e r s ) . U n l i k e P C R , t h e r e i s n o e x t e n s i o n s t e p b e t w e e n
a n n e a l i n g a n d d e n a t u r a t i o n . I n LC R, t h i s c y c l in g p a t t e r n i s r e p e a t e d b e t w e e n
1 0 a n d 3 0 ti m e s , b u t t h e n u m b e r o f cy c le s h a s t o b e o p t i m i z e d f o r ea c h a s s ay .
I n G- LC R , b e t w e e n 3 0 a n d 6 0 c y cl es w i t h d e n a t u r a t i o n a t 8 5 ~ a n d a n n e a l i n g
a t 5 0 - 5 3 ~ h a v e b e e n u s e d . ( 22 '2 3) P r o t o c o l s f o r LC R , p L C R , a n d G - L C R a r e
o u t l i n e d i n T a b l e 1 .
A N A D - r e q u i r i n g t h e r m o s t a b l e l i g a se (2 6'2 8) i s m o s t o f t e n u s e d i n l i g as e -
b a s e d a m p l i f i c a t i o n m e t h o d s . R e c e n t ly , a n o t h e r t h e r m o s t a b l e l ig a se , w h i c h
r e q u i r e s A T P as a c o f a c t o r , h a s b e e n c l o n e d a n d s e q u e n c e d . (29 ) H o w e v e r , t h e
u s e o f t h i s e n z y m e i n D N A a m p l i f i c a t i o n m e t h o d s h a s n o t y e t b e e n e x p l o r e d .
DETE TION M E T H O D S FOR L R PRODU TS
D e t e c t i o n o f t h e L C R p r o d u c t , t h a t i s, t h e t w o l i g a t e d p r i m e r s , w a s i n i t i a l l y
TABLE 2 Noise-to-s ignal ra t io for certa in m ism atch es in the LCR
Oligonucleotide base-target base Noise-to-signal ratioa ( )
A-A, T-T 1.1
T-T, A-A
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M a n u a l S u p p lem e n t lU lm m
accomplished by using a 3 p radioactive label on the 3 end of the upstr eam
primer. The separation of LCR products and primers was achieved by dena-
turing gel electrophoresis, and the LCR product was detected by autoradiog-
raphy. The level of sensitivity reached in an LCR with this detection method
is on the order of 200 target DNA molecules. (4) Winn- Dee n and Iovan nisc i (27)
described a nonisotopic detection method using fluorescently labeled prim-
ers. Detection of the LCR product was accomplished using a fluorescent DNA
sequencer in conjunction with a GENESCANNER (Applied Biosystems). One
of the advantages of this method is that it is relatively easy to quantitate the
amo unt of the LCR products. Furthermor e, each of the primers can be labeled
with a different fluorescent dye to allow unambiguous assignment of ligation
products; incorrect ligation products could be identified by their deviation
from the appropriate color com binati ons. (27) The fluorescent detect ion sys-
tem allows multiplexing with the LCR primers specific for a given mutation
labeled with different fluorescent tags or with the same fluorescent label and
different-sized LCR products./3~ Currently , this me th od is limi ted by the
requirement for sophisticated equipment. An alternative approach for the
nonisot opic detection uses one digoxigenin-labeled primer; the LCR products
are detected in a Southern blot format after gel elect roph oret ic separa tion. (24)
Recently, more convenient methods for the detection of LCR products in
micr oti ter plates have been developed. (31 32) In this format, one LCR pri mer of
a pair is labeled with biotin at the 5 end, whereas the other p rimer is labeled
with a nonisotopi c reporter at the 3 end. Reporter groups tested so far in-
clude a fluorescein dye in blue (FAM, 5-carboxyfluorescein) and digoxigenin.
Direct detection of FAM-labeled LCR products by solution fluorometry
showed poor sensitivity, whereas the use of digoxigenin reporter in conjunc-
tion with anti-digoxigenin antibodies coupled to alkaline phosphatase (AP)
greatly improved the sensitivity. Subsequent detection of the AP could be
achieved using colorimetric, fluorescent, or luminogenic substrates. Winn-
Deen et al. f31) reported that the lumi nogen ic substrate Lumipho s 530 gave the
highest sensitivity in a microtiter plate assay. This sensitivity was only 10-fold
less than with detection methods using radioisotopes or a fluorescent DNA
sequencer. Another nonisotopic detection method for LCR products has been
repor ted by Zebala and Barany. ~33) They ut ilized primer pairs in whi ch one
primer was labeled with a poly(dA) tail at the 5 end whereas the 3 end of the
other primer was tagged with biotin. The ligated products were capture d from
the solution via hybridization of their poly(dA) tails with poly(dT)-coated
paramagnetic iron beads and subsequent magnetic separation. Only the cap-
tured LCR products will carry a S -coupled biot in molecule, whi ch can be
detected with a streptavidin-AP conjugate and a colorimetric substrate.
For the detection of the products from G-LCR, two different meth ods have
been described. Radioactively labeled nucleotides were used to fill in the gap
between the primers, so that the G-LCR products can be det ected by autora-
diog raph y after gel electro phoresis. r Alternatively, the prime rs can be end-
labeled with radioiso topes as described for LCR primers. ~2t) Noni sotopic de-
tection of G-LCR products was achieved by using pairs of primers labeled with
biotin or fluorescein, respectively. Ligated oligonucleotides were capt ured o n
antifluorescei n-coated microparticles and detected with an antibiotin-AP con-
jugate. AP activity was subsequently detected with the fluorescent substrate
methy lumb elli fero ne phosphate. (23)
C U R R E N T P P L I C T I O N S O F L C R
L R assays have been develop ed for the dete ct ion of ge net ic d iseases as we l l
as for the detection of bacteria and viruses. An overview of the current ap-
plications of LCR is shown in Table 3. In many of these applications, LCR is
5 8 P CR M e t h o d s a n d A p p l i c a t i o n s
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l l | i l l l a n u a i S u p p lem e n
T B L E
3 C ur r e n t A pp l i ca t i ons o f L C R and G - LC R
Target Format Reference
G e n e t i c d i s e a s e s
[3-sickle cell
hemoglobinemia
13-sickle ce ll
hemoglobinemia
Cystic fibrosis
Cystic fibrosis
Leber 's h ereditary optic
neuropathy
Hyperkalemic periodic
paralysis
Bovine leukocyte
adhesion deficiency
acteria
Borrelia burgdorferi
Listeria monocytogenes
Neisseria gonorrhoeae
LCR, isotopic
LCR, fluoresce nt
PCR-LDR, fluorescent
LCR and G-LCR, isotopic
PCR-LCR, nonisotopic
PCR-LCR, fluorescent
PCR-LCR, nonisotopic
LCR, nonisotopic
PCR-LCR, nonisotopic
G-LCR, non isotop ic
Barany~4)
W inn-D een an d Iov annisci ~27)
Egge rding et al. Cts)
W inn -De en et al. ~16~
Fang et al. r
Zebala and Barany
Fe ero et al. ~3~
W ang et al. r
Batt et al. r
Hu et al. 43)
Wiedmann e t a l . 2 ~
Birkenmeyer and M ms tron g (2s)
Erwinia stewartii
Mycobacterium
tuberculosis
Chlamydia trachomatis
V i r u s e s
Hum an papillomavirus
Herpes simplex virus
HIV DNA
O t h e r t a r g e t s
Ha ras protooncogene
Ha ras
protooncogene
G-6-PD
HOXB7
PCR-LCR, isotopic
LCR, fluoresce nt
G-LCR, isotopic
LCR, nonisotopic
LCR, nonisotopic
LCR, nonisotopic
LCR, nonisotopic
PCR-LCR
RT-PCR-LDR, isotopic
RT-PCR-LCR, isotopic
Wilson et a l . 4 ~
lovan nisci and W inn-D een ~42)
Dille et al. ~22)
Bond et al . 46)
Rineh ardt et al. (45)
Carrino and
L a f f l e r 4 4 )
K~ilin et a l J 2 4 )
Wei et al . 48)
Prchal et a l . 0 4 )
Ch ari ot et al. ~49~
p r e c e d e d b y a n i n i ti a l P C R s te p t o a c h i e v e a g r e a te r s e n s i t i v i t y o f t h e r e s p e c -
t i v e a s s a y s .
D e t e c t i o n o f G e n e t i c D i s e as e s
I n t h e i n i ti a l p u b l i s h e d r e p o r t s d e s c r i b i n g LC R, d i s c r i m i n a t i o n b e t w e e n n o r -
m a l 13A- a n d s ic k l e [3 S -g lo b in g e n o t y p e s i n h u m a n s w a s a c h i e v e d u s i n g e i t h e r
a n i s o t o p i c d e t e c t i o n m e t h o d (4) o r f l u o r e s c e i n - l a b e l e d L C R p r i m e r s . (z7) T w o
s e ts o f L C R p r i m e r s w e r e u s e d , o n e s p e c i f ic f o r t h e n o r m a l a l le l e a n d t h e o t h e r
s p e c if i c f o r t h e m u t a t i o n . T h e s e t w o p r i m e r s e ts w e r e a p p l i e d i n t w o s e p a r a t e
L C R r e a c t i o n s , a n d t h e L C R p r o d u c t s w e r e a n a l y z e d s e p a r a t e l y . T h i s d e s i g n
a l lo w s e a s y id e n t i f i c a t i o n o f h o m o z y g o u s a s w e l l a s o f h e t e r o z y g o u s c a r r i e rs
o f t h e a l l e l e s o f i n t e r e s t .
R e c e n tl y , L CR h as b e e n e x p l o i te d f o r t h e d e t e c t i o n o f o t h e r m u t a t i o n s
r e s p o n s i b l e f or g e n e t ic d i s o r d e rs i n h u m a n s a n d a n i m a l s . E x a m p l e s i n c l u d e
c y s t i c f i b r o s i s , ~34) L e b e r ' s h e r e d i t a r y n e u r o p a t h y , (33~ a n d h y p e r k a l e m i c p e r i -
o d i c p a r a ly s is 13~ i n h u m a n s a n d b o v i n e l e u k o c y t e a d h e s i o n d e f i c i e n c y
( B LA D ) ~2s) i n c a t t l e . S c r e e n i n g f o r t h e A F 5 0 8 , W 1 2 8 2 X , a n d o t h e r c y s t i c f i b r o -
s is m u t a t i o n s w a s p e r f o r m e d e i t h e r i n tw o s e p a r a t e LC R r e a c t i o n s t a r g e t i n g
t h e n o r m a l a n d m u t a n t a ll el e o r i n a c o m p e t i t i v e r e a c t i o n w i t h si x p r i m e r s ,
i n c l u d i n g t w o c o m m o n p r i m e r s, tw o f o r t h e m u t a n t , a n d t w o f or t h e n o r m a l
a l le l e . (34 ) D e t e c t i o n o f a l le l e s l e a d i n g t o h y p e r k a l e m i c p e r i o d i c p a r a l y s i s w a s
a c h i e v e d b y u s i n g a m u l t i p l e x P C R - c o u p l e d L CR s i m u l t a n e o u s l y t a r g e t i n g
t h r e e d i f f e r e n t p o t e n t i a l s i n g l e b a s e - p a i r m u t a t i o n s J 3~ F o r a ll o f t h e s e m u -
P CR M e t h o d s a n d A p p l i c a t i o n s 5 9
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M a n u a l S u p p l e m e n t II I I I
tations, LCR primers for the mutant and for the normal allele were included
in th e LCR, there fore screen ing for each of thes e alleles. LCR primers were
labeled using a fluorescent dye (FAM) and primers of different length s so that
LCR products for the various mutati ons and alleles could be differentiat ed on
a fluorescent DNA sequencer by their relative mobility. This approach has
recently been extended to the detection of different mutations causing cystic
fibrosis. (ls 16) Detect ion of tri nucleoti de repeats that can give rise to cert ain
diseases, including myotonic dystrophy, has been acheived using repeat ex-
pansi on det ection (RED). In this format, tr inucleot ide rep eat-co ntaini ng oli-
gonucleotides are ligated when bound in tandem to the target and by cycling
greater l engths of these ligatio n pr oducts are generat ed. ~36)
D e t e c t i o n o f a c t e r ia l P a t h o g e n s
Given the potenti al of LCR, attempts were made to use this te chni que for the
identification and detection of bacterial pathogens. Detection systems for
bacteria based on PCR or other molecular biology techniques usually depend
on the availability of well-characterized genus- or species-specific target
genes. This strategy is easily applied to extensively documented bacterial
pathogens, where the sequence of one or more genes is known. However, for
many plant and animal pathogens as well as nonpathogenic bacteria from
environmental sources, often there is not sufficient information available to
design species-specific PCR primers. The 16S rDNA, encoding part of the
ribosomal RNA, consists of both highly conserved and variable regions, the
latter usually containing at least single base pair differences that are species-
specific. A general met hod for PCR amplificat ion and sequenci ng of this gene
has b een described by Weis burg et al. ~37) Our group initial ly util ized these
techni ques to sequence the 16S rDNA gene of different isolates of the h um an
pathogen
L. monocy togenes
and the closely related nonpath ogenic bacterium
L. innocua. 38) This method was preferred over direct sequencing of the 16S
rRNA using reverse transcriptase, which is not precise enoug h to identif y all
nucleotides accurately. (39) After identifying cons ist ent single base-pair differ-
ences specific for L. monocytogenes, LCR primers were designed to identify this
bacterium based on one of these differences. To improve the sensitivity of
this LCR, we further emp loye d a set of flankin g PCR primer s to ampl ify
initially the segment conta ining the specific single base-pair difference. (2~
This PCR-coupled LCR was shown to be highly specific for
L. monocy togenes
and was able to detect, at a minimum, l0 colony-forming units of L. mono-
cytogenes using a noni soto pic dete ctio n meth od. (32)
The same approach was used to develop an LCR-based detection method
for the plant pathogen Erw in ia s tew ar t i i . 4~ After sequencing parts of the
16S rDNA gene of E. s tewarti i and the closely related saprophyte E. herbicola,
E . s t e w a r t i i - s p e c i f i c single base-pair differences were identified. These were
again used to design LCR primers for a PCR-coupled LCR, which proved to be
specific for
E. s tewarti i .
The dev elopm ent of these two PCR-coupled LCR assays for the detecti on of
L. monocy togenes
and
E. s tew ar t i i
suggests that this system is generally appli-
cable for the develo pment of a sensitive detecti on assay for all bacteria when
little or no prior genetic information is available.
Application of LCR for the detection of bacterial pathogens is not limited
to targets wi thi n t he rDNA. Iova nnisci and Win n-D een (42) utili zed LCR to
detect
M y c o b a c t e r i u m t u b e r c u l o s i s
DNA, based on the inserti on seque nce
IS6110, which is specific for this important pathogen. Using fluorescently
labeled prime rs (ROX, TAMARA, FAM, JOE) and a fluores cent DNA sequ encer,
it was possible to detect as few as 100 copies of the target molecule even in the
presence of unrelat ed DNA. Furthermore, a nonisotopi c LCR for the det ection
of Borrelia burgdorferi has bee n described. (43)
6 0 P C R M e t h o d s a n d A p p l i c a t i o n s
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l III IM a n u a l S u p p lem e n
Assays for the detection of the bacterial path ogen s
Neisseria gonorrhoeae
and
Chlamydia trachomatis
using G-LCR have also been described. (22 23) These
assays are based on 2-bp differences between the target bacterium and closely
related nonpathogenic bacteria. The sensitivity of these assays is approxi-
mately one
N. gonorrhoeae
cell using the nonisotopic detection method and
three
C. trachomatis
elementary bodies using an isotopic detection method
with electrophoretic separation of the products from unligated primers. De-
tection of
N. gonorrhoeae
was achieved by using G-LCR probes target ing se-
quences in the gene coding for the cell-surface opacity (Opa) protein or in the
gene for the pilin proteins. ~23~ The targeted sequences show on ly 2-bp differ-
ences between N. gonorrhoeae and the closely related Neisseria rneningitidis
whic h is sufficient for clear diffe rentiati on by G-LCR. For the specific detec-
tion of C. trachomatis primers were used that recognized species-specific se-
quences either in the gene for the major outer membrane protein or on a
cryptic plasmid322~
e t e c t i o n o f V i r u s e s
Only preliminary reports on the use of LCR for the identification and/or
detection of viruses have been published. A nonisotopic ligase-based DNA
amplification assay using oligonucleotides targeting part of the gag region of
HIV-1 has been described. The sensitivity of this assay is between 5 and 10
HIV-1 molecules, which is comparable to the level of sensitivity reached by
PCR344) LCR technology has also been applied for the nonradioactive detec-
tion of herpes simplex virus and HPV and allowed rapid detection of these
viruses as compared to traditional detection methods using cell culture tech-
niques . ~4s,46)
Another ligase-mediated approach for detection of HIV used Qf3-replicase
to amplify a target-dependent ligation product of amplifiable hybridization
probes. This strategy helps to overcome the problem of target-independent
amplif ica tion of no nhybrid ized probes in Q[3 replicase assays. (47)
etect ion o f Oth er Target Sequences
K~ilin et al. (24) described the evaluation of LCR for the det ect ion of sing le
base-pair mutations in the
Ha-ras
proto-oncogene. This group reported a sen-
sitivity of 250 molecules for the targeted mutation but could not differentiate
the mutant from the normal allele when a 1:100 ratio of mutant to normal
DNA was used. These problems might be caused by the use of LCR primers
with a blunt end rather than a single base-pair overhang, which is known to
cause higher target-independent ligation (see above, under Design of LCR
Primers). Wei et al., (48~ on the other hand, were successful in developin g a
combination of PCR and LCR for the detection of point mutations in the
Ha-ras
proto-oncogene. Using two cycles of
M spI
restriction, PCR amplifica-
tion, and a subsequent LCR amplification, they were able to detect mutant in
a background of 108 wild-type alleles.
Prchal et al. (14) used a combinat ion of RT-PCR and LDR for transcriptional
analysis to determine the active X-chromosome based on a polymorphic
locus on this chromosome. In the first step, DNA isolated from a person is
tested for heterozygosi ty in the target allele using a PCR-coupled LDR. Only
persons found to be heterozygotic are then subjected to transcriptional anal-
ysis. For this purpose mRNA is isolated from the cells of interest, for example,
lymphocytes, myeloid cells, and fibroblasts, and used for RT-PCR amplifica-
tion and subsequen t LDR. The LDR then detects the allele transcribed in the
isolated cells, therefore indicating the clonality of these cells. This assay can
be applied for the specific and sensitive determination of clonality in cells,
P C R M e t h o d s a n d A p p l i c a t io n s 6 1
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M a n u a l S u p p l em e n t m w
cell linages, and tissues, which is important for studies of neoplastic disorders
and embryologic development.
Another application of a cancer-related mutat ion using a RT-PCR-coupled
LCR has been described by Chario t et al. (49) This group used LCR to detec t th e
expression of stop codon polymorphism in the homeo domain sequence
HOXB7 of a breast cancer-derived cell line. This work demonstrates the po-
tential of RT-PCR-coupled LCR in RNA diagnost ic procedures by the example
of the sensitive detection of single-base polymorphisms in rare mRNA tran-
scripts.
OUTLOO
With the continuing emergence of sequence data for the human genome as
well as the genomes of other species (e.g., bovine, equine), the potential of
LCR to detect genetic diseases that result from single base-pair mutations is
immense. One of the inherent advantages of LCR is its potential for automa-
tion. The LCR product consists of two covalently joined primers that can be
easily detected using different enzyme-linked or direct fluorescent labels.
Formatting of multiplex LCR assays will further improve screening samples
for an array of different single base-pair changes in a single tube. Automated,
multip lex LCR or PCR-coupled LDR/LCR assays have a variety of po tenti al
applications, such as ~s) (1) screening of large popula tions for monogenic
disease polymorphisms; (2) determining HLA haplotypes in tissue typing, for
example, for transplantation; and (3) screening for multiple bacterial species
after a generic PCR amplification of 16S rDNA sequences.
In clinical diagnosis of pathogenic bacteria and viruses, the specificity of
LCR could be useful in many applications. The detection of single base-pair
differences in bacterial pathogens may be valuable with respect to antibiotic
resistance arising from point mutations, for example, in some cases of mac-
rolide resistance ~s~ or from tra nsformational exchange as occurs in sensitive
and resistant strains, for example, in N. m e n in g i t i d i s . Csl In viral pathogens,
the identification of subpopulations with genetic differences may be impor-
tant with regard to host range, virulence characteristics, and drug resistance.
Furthermore, the application of LCR and PCR-coupled LCR assays for the
detection of specific bacteria based on at least a single base-pair difference in
the 16S rDNA gene has great potential. As outlined above, such a system
circumvents the need to identify species-specific genes, as warranted for PCR
or other nucleic acid-based assays. With emerging interest in yet poorly char-
acterized bacteria, this method should have a great potential as a detection
system.
C K N OW L E D G M E N T S
We are grateful to many of our colleagues, including A. Beaudet, L. Birken-
meyer, E.P. Hoffman, U. Landegren, T. Uchida, V.L. Wilson, and E.S. Winn-
Deen, who provided manuscripts in preparation, submitted, or in press, and
reprints. Part of the work presented here was supported by the Northeast
Dairy Foods Research Center (to C.A.B.), Eastern Artificial Insemination (to
C.A.B.), a grant from the Cornell Center of Advanced Techn ology (CAT) in
Biotechnology (which is sponsored by the New York State Science and Tech-
nology Foundation, a consortium of industries and the National Science
Foundation) (to C.A.B.), a grant from Applied Biosystems Division of Perkin-
Elmer (to F.B.), and the National Institutes of Health (GM 41337-03) (to F.B.).
M.W. was supported by a stipend of the Gottlieb Daimler- und Carl Benz-
Stiftung (2.92.04). W.W. s work was supported by a grant from the New York
State Sweet Corn Research Association (to H.R. Dillard).
6 2 P C R M e t h o d s a n d A p p l i c a t io n s
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l l | l l l l a n u a l S u p p l em e n
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