Nucleic Acids Research Advance Access first published online on January 7, 2009
This version published online on February 8, 2009
Nucleic Acids Research, doi:10.1093/nar/gkn1024
© 2009 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction
Qiuping Guo,
Xiaohai Yang,
Kemin Wang*,
Weihong Tan,
Wei Li,
Hongxing Tang and
Huimin Li
State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Biomedical Engineering Center, Institute of Biological Technology, Key Laboratory for Bio-Nanotechnology and Molecular Engineering of Hunan Province, College of Material Science and Engineering, Hunan University, Changsha 410082, P.R. China
*To whom correspondence should be addressed. Tel/Fax: +86 731 8821566; Email: kmwang{at}hnu.cn
Received May 6, 2008. Revised December 6, 2008. Accepted December 9, 2008.
 |
ABSTRACT
|
|---|
Here we have developed a sensitive DNA amplified detection method
based on isothermal strand-displacement polymerization reaction.
This method takes advantage of both the hybridization property
of DNA and the strand-displacement property of polymerase. Importantly,
we demonstrate that our method produces a circular polymerization
reaction activated by the target, which essentially allows it
to self-detect. Functionally, this DNA system consists of a
hairpin fluorescence probe, a short primer and polymerase. Upon
recognition and hybridization with the target ssDNA, the stem
of the hairpin probe is opened, after which the opened probe
anneals with the primer and triggers the polymerization reaction.
During this process of the polymerization reaction, a complementary
DNA is synthesized and the hybridized target is displaced. Finally,
the displaced target recognizes and hybridizes with another
probe, triggering the next round of polymerization reaction,
reaching a target detection limit of 6.4
x 10
–15 M.
|
INTRODUCTION
|
|---|
Rapid growth of available sequence data has made the detection
of nucleic acids critical to the development of modern life
sciences (
1–3). Among the many methods devised for the
detection and analysis of nucleic acids, amplification is one
of the most important concepts since it permit the highest analytical
sensitivity (
4–8). PCR provides a general protocol for
the amplified detection of DNA. Although the PCR method is time
consuming and not free of limitations, it provides the most
versatile method to detect minute amounts of DNA. The design
of alternative approaches for the sensitive detection of DNA
is in continuous demand. Enzyme conjugates (
4,
5), DNAzymes (
6)
and nanoparticles (
7,
8) have been used as amplifying labels
for biorecognition events. Recently, a means of amplified DNA
detection methods have been developed based on scission or replication.
Since these DNA detection systems can be operated autonomously
and repeatedly while they are trigged by the target, large amounts
of DNA products are yielded to enhance the signal, the sensitivity
of DNA detection is thereby significantly increased (
9–14).
Several systems have been designed to construct high sensitive
DNA detection methods, e.g. autonomous replication of DNA/FokI
cutter units (
9), autonomous polymerization of a peroxidase-mimicking
DNAzyme (
10) and autonomous aggregation of Au nanoparticles
(
11).
Here we report a method for amplified detection of DNA based on the inherent signal-transduction mechanism of the hairpin fluorescence probe and strand-displacement property of polymerase. As such, the hairpin fluorescence probe acts as a template of polymerization reaction and fluorescence signal carrier, while the target acts as a trigger of polymerization reaction. The activation of this DNA detection system is based on the conformational change of the probe induced by hybridization between probe and target DNA. In this method, the target DNA is displaced in the process of the polymerization reaction and then hybridized to another probe. Thus, in essence, this design method allows hybridization, polymerization reaction and displacement to occur cycle-after-cycle, producing, at the same time, an amplified fluorescent signal sufficient to indicate the presence of trace amount of target DNA. Even though, this method is based on a simple design and is easy to use, it has demonstrated a high magnitude of amplified, sensitive detection with a limit of 6.4 x 10–15 M.
|
MATERIALS AND METHODS
|
|---|
Materials
The hairpin fluorescence probe and oligonucleotide were commercially
synthesized by TaKaRa Bio Inc. (Dalian, China). Sequences of
the oligos are listed in
Table 1. The polymerase Klenow fragment
exo
– was purchased from New England Biolabs, Inc. The
deoxynucleotide solution mixture (dNTPs) was purchased from
TaKaRa Bio Inc. (Dalian, China) and the DMSO was obtained from
Sigma. All other reagents were of analytical grade. Deionized
water was obtained through the Nanopure Infinity
TM ultrapure
water system (Barnstead/Thermolyne Corp., Dubuque, IA, USA).
Fluorescence measurement
All fluorescence measurements were carried out on a F2500 fluorometer
(Hitachi, Japan) equipped with an aqueous thermostat (Amersham)
accurate to 0.1°C. Excitation and emission wavelengths were
set at 496 and 517 nm, respectively, with 5-nm bandwidths. The
emission spectra were obtained by exciting the samples at 490
nm and scanning the emission from 500 to 600 nm in steps of
1 nm. All samples were incubated at 37°C. When the fluorescence
intensity became steady, the target was added into the mixture,
and the fluorescence intensity was recorded simultaneously.
Target hybridization with hairpin fluorescence probe
Three samples were prepared to identify the hybridization feature of the hairpin fluorescence probe: sample A containing 5.0 x 10–8 M probe only; sample B containing 5.0 x 10–8 M probe and 5.0 x 10–8 M target; sample C containing 5.0 x 10–8 M probe and 1.0 x 10–7 M target. All samples were performed in 80 µl solution containing 50 mM Tris–HCl (pH 8.0) and 5 mM MgCl2 and incubated at 37°C. The difference of fluorescence intensity between samples with and without target indicated the detection capability of the hairpin probe.
The temperature melting curve of the hairpin fluorescence probe and its target
Two samples were prepared to determine the thermal profiles of the hairpin fluorescence probe and its target: Sample A containing 5.0 x 10–8 M probe only and sample B containing 5.0 x 10–8 M probe and 5.0 x 10–8 M target. All samples were performed in 80 µl solution containing 50 mM Tris–HCl (pH 8.0) and 5 mM MgCl2. The temperature was increased from 40°C to 90°C in steps of 2°C, with each step lasting 2 min.
Amplified detection of target
The experiments were performed in 80 µl solution consisting of 5.0 x 10–8 M probe, 5.0 x 10–8 M primer, 15 U polymerase Klenow fragment exo–, 100 µM dNTPs, 6% DMSO, 1 mM DTT and 5 mM MgCl2 in 50 mM Tris–HCl (pH 8.0) and incubated at 37°C. A series of targets at different concentrations from 1.0 x 10–10 M to 1.28 x 10–15 M were then added to the mixture solution and the fluorescence intensities were recorded.
Gel electrophoresis
A 20% non-denaturing PAGE analysis of the products by the isothermal strand-displacement polymerization reaction was carried out in 1 x TBE (pH = 8.3) at 80 V constant voltage for about 3 h. After Sybr green I staining, gels were scanned using an Image Master VDS-CL (Amersham Biosciences).
|
RESULTS AND DISCUSSION
|
|---|
Principle of the amplified DNA detection method
The principle of our isothermal amplified detection DNA method
is shown in
Figure 1. This DNA detection system consists of
a hairpin fluorescence probe, a short primer and polymerase.
The hairpin fluorescence probe possesses a stem-loop structure
with a fluorophore and a quencher linked to the ends of the
stem. The stem is 11-nt sequences long, and the loop is complementary
to the target. The primer is an 8-nt sequences long, which is
complementary to the stem region of the probe at 3'-end. In
the absence of a target, the stem-loop conformational probe
is unable to anneal with the primer to induce a polymerization
reaction. However, in the presence of target DNA, the probe
recognizes and hybridizes with it and undergoes a conformational
change, leading to stem separation (Step 1). Following this,
the primer anneals with the open stem and triggers a polymerization
reaction in the presence of dNTPs/polymerase (Step 2). Next,
in the process of primer extension, the target is displaced
by the polymerase with strand-displacement activity, after which
a complementary DNA is synthesized, forming a probe–cDNA
complex (Step 3). Finally, to renew the cycle, the displaced
target hybridizes with another probe, which triggers yet another
polymerization reaction (Step 4). Throughout this cyclical process,
the hairpin fluorescence probe plays a key role as both template
of polymerization reaction and fluorescence signal carrier,
while the target acts as a trigger of polymerization reaction.
Amplified detection results from this activity because the target
can be displaced and trigger the polymerization reaction circularly.
Under these conditions, even minute amounts of targets can produce
obvious fluorescence enhancement based on the circular polymerization
reaction triggered by the displaced targets themselves. Therefore,
by monitoring the increase of fluorescence intensities, we could
detect the target with high sensitivity.
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 1 The mechanism of isothermal amplified detection of DNA based on strand-displacement polymerization reaction. In the presence of target DNA, the hairpin fluorescence probe recognizes and hybridizes with it and undergoes a conformational change, leading to stem separation (Step 1). Following this, the primer anneals with the open stem of the hairpin fluorescence probe and triggers a polymerization reaction in the presence of dNTP/polymerase (Step 2). Next, in the process of primer extension, the target is displaced by the polymerase with strand-displacement activity, after which a complementary DNA is synthesized, forming a probe–cDNA complex (Step 3). Finally, to renew the cycle, the displaced target hybridizes with another hairpin fluorescence probe, which triggers yet another polymerization reaction (Step 4).
|
|
Design of the hairpin fluorescence probe
To achieve the degree of amplification desired, our design relies
upon the annealing of the primer with the open stem after the
hairpin fluorescence probe hybridizes with the target. Traditional
hairpin probes, such as molecular beacons (MBs) contain 5–8-nt-long
stem sequences and 15–35-nt-long loop sequences, which
undergo conformational change upon hybridization with the target
(
15,
16). In contrast, the hairpin fluorescence probe used in
this study has a stem long enough to ensure that stem hybridization
affinity will be stronger than hybridization affinity with the
primer. Therefore, in the absence of target, the primer does
not induce polymerization reaction. On the other hand, a stem
that is too long would restrain hairpin probe conformational
change upon hybridization with target. Hence, in this work,
the stem consisted of 11-nt-long sequences, thus forming a structure
stable enough to prevent the primer from annealing with duplex
stem. In order to open long stem hairpin probe upon hybridization
with target, a ‘shared-stem’ (
17) structure is designed,
i.e. besides the loop region, six bases of the stem at 5'-end
is complementary to the target (
Table 1). ‘Shared-stem’
structure is a design variant of conventional MB where one arm
of the stem participates in either hairpin formation or target
hybridization. The hybrid of shared-stem MB with its target
has more stable structure than the hybrid of conventional MB
with its target (
17).
Detection capability of the hairpin fluorescence probe
The activation of the DNA system is based on the conformational change of the hairpin fluorescence probe upon hybridization with target DNA. The enhancement of fluorescence intensities, as shown in Figure 2, reveals the conformational change of hairpin fluorescence probe. Curves a–c are fluorescence intensities of 5.0 x 10–8 M probe in the presence of 0, 5.0 x 10–8 and 1.0 x 10–7 M target, respectively. The signal-to-background ratio (SBR) is often used to characterize the detection capability of the hairpin fluorescence probe and is calculated as (Fopen – Fbuffer)/(Fclosed – Fbuffer), where Fbuffer is the fluorescence intensity of buffer solution, Fclosed is the fluorescence intensity of buffer solution by adding probe and Fopen is the fluorescence intensity of buffer solution by adding probe and target (15,16). In this study, SBR of the hairpin fluorescence probe is 39.5 and 45.2, when the concentration of target is 5.0 x 10–8 and 1.0 x 10–7 M, respectively. These results implied that the hairpin fluorescence probe underwent a conformational change upon hybridization with the target and that fluorescence was restored. Therefore, the hairpin fluorescence probe was suitable to act as a template for polymerization reaction in this study.
View larger version (16K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 2 Hairpin fluorescence probe hybridization with target. The mixture containing 5.0 x 10–8 M probe with 0 M (curve a), 5.0 x 10–8 M (curve b) and 1.0 x 10–7 M (curve c) target, respectively. All samples were incubated at 37°C.
|
|
Thermal profiles of the hairpin fluorescence probe (curve a)
and the hybrid with its target (curve b) are shown in
Figure 3.
The
Tm value for probe and the probe hybridization to its target
was 83°C and 56°C, respectively that are higher than
the temperature of the polymerization reaction. This result
indicated that the hairpin fluorescence probe, as designed,
can be used in this strategy.
View larger version (9K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 3. Thermal profiles of hairpin fluorescence probe and the hybrid with its target. The mixture containing 5.0 x 10–8 M probe with 0 M (curve a) and 5.0 x 10–8 M (curve b) target, respectively. The temperature was increased from 40°C to 90°C in increment of 2°C, with each increment lasting 2 min.
|
|
Verification of the amplified DNA detection method
As previously described, the hairpin fluorescence probe undergoes
a conformational change upon hybridization with its target and
provides an opened stem for primer annealing. Strand-displacement
polymerization reaction is then triggered in the presence of
polymerase. Having proved the suitability of the hairpin probe,
as designed, we now investigated the feasibility of this method
for amplification of DNA detection. As shown in
Figure 4, the
fluorescence intensity increased upon addition of target to
the mixture containing dNTPs and polymerase (curve a), indicating
that the polymerization reaction was triggered by the target.
Furthermore, the fluorescence intensity maintained its increase
with time, indicating that the continuous formation of probe–cDNA
complex was the result of circular polymerization reaction.
The fluorescence intensity of the solution reached maximum within
4000 s, indicating that the target quantitatively converted
the probe to the signaling state. In the absence of a target,
no fluorescence intensity change was observed, indicating that
no polymerization reaction was triggered (curve b). A control
experiment performed with addition of random single-stranded
DNA revealed that polymerization reaction was not triggered
even in the presence of dNTPs/polymerase (curve c), implying
that the detection was target-specific.
View larger version (13K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 4. Verification of isothermal strand-displacement polymerization reaction. The mixture containing 5.0 x 10–8 M probe and 5.0 x 10–8 M primer with 6.25 x 10–9 M target (curve a), 6.25 x 10–9 M random DNA (curve c) added at t0 in the presence of dNTPs/polymerase respectively; curve b: no target added.
|
|
The above results were further confirmed by electrophoresis.
Figure 5 shows electrophoresis results of products synthesized
by polymerization reaction at various reaction time intervals.
Lanes a, b and c are nucleic acid bands generated at 60, 20
and 0 min, respectively. As expected, the content of probe–cDNA
complex (the middle band in lane b) increased as the reaction
time increased. In addition, a new product (the upper band)
with a slower migration speed than that of probe–cDNA
complex appeared in lane b. As shown in
Figure 1, an intermediate
product of probe–target–cDNA complex exists in the
process of primer extension before target was displaced (Step
2). Therefore, the new band might be the probe–target–cDNA
complex.
View larger version (38K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 5. Non-denaturing PAGE analysis of the products by the isothermal strand-displacement polymerization reaction. a–c curves: products generated by the isothermal strand-displacement polymerization reaction containing 2.0 x 10–7 M probe, 2.0 x 10–7 M primer and 2.0 x 10–8 M target at different time intervals in the presence of dNTPs/polymerase, (a) t = 60, (b) t = 20, (c) t = 0 min. All samples were incubated at 37°C.
|
|
Amplified detection of target with high sensitivity
Figure 6A shows the fluorescence intensities observed upon analyzing
different concentrations of targets with this method. The results
showed that the number of opened probes increased as the concentration
of the targets increased.
Figure 6B shows the relationship between
the rate of fluorescence enhancement (for 1000 s) and the concentration
of target. As the concentration of the target increases, the
rate of fluorescence enhancement increases. For this method,
when the target is 6.4
x 10
–15 and 1.28
x 10
–15 M, the (F – F
0) is 10.3 and 8.7, the background noise
is 3.2, and then the signal-to-noise is 3.2 and 2.7 (F
0 is the
fluorescence intensity of the solution before the polymerization
reaction, and F is the fluorescence intensity of the solution
after the polymerization reaction for 1000 s). Therefore, 6.4
x 10
–15 M was considered as the detection limit, which
is two or three order of magnitude lower than that of other
reported DNA amplified detection methods (
10,
11). The turnover
rate of this method is also calculated. As shown in
Table S-2,
the turnover rate (s
–1) of this method was 0.051, 0.211,
0.820 and 2.828, with target concentration of 8.0
x 10
–13,
1.6
x 10
–13, 3.2
x 10
–14 and 6.4
x 10
–15 M,
respectively. Since the rate of fluorescence enhancement is
influenced by multiple factors, including the concentration
of input target and reaction time, the turnover rate of this
method varies according to different target concentrations.
View larger version (23K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 6. Detection of different concentrations of target based on isothermal strand-displacement polymerization reaction. Experiments were performed in the presence of 15U polymerase Klenow fragment exo– and 100 µM dNTPs with 5 x 10–8 M probe, 5 x 10–8 M primer, and different concentrations of target. (A) Monitoring the fluorescence intensity of this amplified DNA detection method over a range of target DNA concentrations. The curves from a to i contain the target with 1.0 x 10–10, 2.0 x 10–11, 4.0 x 10–12, 8.0 x 10–13, 1.6 x 10–13, 3.2 x 10–14, 6.4 x 10–15, 1.28 x 10–15 and 0 M, respectively. All samples were incubated at 37°C. (B) The relationship of the rate of fluorescence enhancement with target DNA concentration.
|
|
In order to confirm whether the high sensitivity of DNA detection
results from the circular strand-displacement polymerization
reaction, control experiments with target at various concentrations
reacting with the hairpin fluorescence probe in the absence
of primer/polymerase were also carried out. As shown in
Figure 7,
fluorescence intensities also increased with increasing concentration
of target, a result obviously produced by the hybridization
of target with probe. However, the fluorescence intensities
reached a plateau within only a few minutes, implying that no
circular strand-displacement polymerization reaction occurred
without the inclusion of both primer and polymerase. The detection
limit was only 1.0
x 10
–10 M.
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
|
Figure 7. The hairpin fluorescence probe response to the target. Experiments were performed in the absence of polymerase and dNTPs with 5 x 10–8 M probe and different concentrations of target. The curves from a to e contain the target with 1.25 x 10–8, 2.5 x 10–9, 5 x 10–10, 1 x 10–10 and 0 M, respectively. All samples were incubated at 37°C.
|
|
Moreover, this method is based on an isothermal amplification
process, and the product of primer extension is 34-nt sequences
long. Consequently only a small interval of time is required
for each primer extension. Therefore, although this method is
a linear amplification, the circular speed of it is much quicker
than that of PCR, and high sensitivity is achieved in a shorter
time. Notwithstanding these advances, this method is still not
as sensitive as PCR, and, as a result, a pretreatment, such
as enrichment, may be necessary for some biological samples.
In our case, however, the probe was hybridized with a single-strand
DNA, indicating that this method can be mainly used for detection
of actual biological samples with single-strand genomic DNA.
In nature, many viruses have single-strand genomic DNA, such
as Parvoviridae, Geminiviruses, Microviridae or Inoviridae (
18).
Therefore, this method has potential application to the analysis
of pathogens.
|
CONCLUSION
|
|---|
In summary, a method with a novel platform which amplifies single-strand
DNA (ss DNA) detection based on polymerase-induced isothermal
strand-displacement polymerization reaction was presented in
this paper. The detection limit of this method is 6.4
x 10
–15 M, which is five orders of magnitude lower than that of the
same hairpin fluorescence probe hybridizing with the target
without primer and polymerase. Therefore, this isothermal and
rapid analysis of target DNA demonstrated the appealing bioanalytical
features of this method, which can be expected to provide a
sensitive platform for amplified detection and subsequent analysis
of nucleic acids.
|
SUPPLEMENTARY DATA
|
|---|
Supplementary Data are available at NAR Online.
|
FUNDING
|
|---|
The National Key Basic Research Program of China (2002CB513110);
the Major International Joint Research Program of Natural Science
Foundation of China (20620120107); the Key Project of Natural
Science Foundation of China (90606003); the China National Key
Projects (2005EP090026); the Hunan Province Natural Science
Foundation of China (08JJ1002). Funding for open access charge:
90606003.
Conflict of interest statement. None declared.
|
REFERENCES
|
|---|
- Willner I, Willner B. Biomaterials integrated with electronic elements: enroute to bioelectronics. Trends Biotechnol. (2001) 19:222–230.[CrossRef][Web of Science][Medline]
- Wang J. Survey and summary: from DNA biosensors to gene chips. Nucleic Acids Res. (2000) 28:3011–3016.[Abstract/Free Full Text]
- Tom NG, Lars R, Oliver S. Triplex molecular beacons as modular probes for DNA detection. Angew. Chem. Int. Ed. (2007) 46:5223–5225.[CrossRef]
- Caruana DJ, Heller A. Enzyme-amplified amperometric detection of hybridization and of a single base pair mutation in an 18-base oligonucleotide on a 7-mm-diameter microelectrode. J. Am. Chem. Soc. (1999) 121:769–774.[CrossRef][Web of Science]
- Patolsky F, Lichtenstein A, Willner I. Detection of single-base DNA mutations by enzyme-amplified electronic transduction. Nat. Biotechnol. (2001) 19:253–257.[CrossRef][Web of Science][Medline]
- Liu J. A colorimetric lead biosensor using DNAzyme-directed assembly of gold nanoparticles. J. Am. Chem. Soc. (2003) 125:6642–6643.[CrossRef][Web of Science][Medline]
- Wang J, Liu G, Merkoc A. Electrochemical coding technology for simultaneous detection of multiple DNA targets. J. Am. Chem. Soc. (2003) 125:3214–3215.[CrossRef][Web of Science][Medline]
- Wang J, Rincon O, Polsky R, Dominguez E. Electrochemical detection of DNA hybridization based on DNA-templated assembly of silver cluster. Electrochem. Commun. (2003) 5:83–86.[CrossRef][Web of Science]
- Weizmann Y, Cheglakov Z, Pavlov V, Willner I. Autonomous fueled mechanical replication of nucleic acid templates for the amplified optical detection of DNA. Angew. Chem. Int. Ed. (2006) 45:2238–2242.[CrossRef]
- Weizmann Y, Beissenhirtz MK, Nowarski ZCR, Kotler M, Willner I. A virus spotlighted by an autonomous DNA machine. Angew. Chem. Int. Ed. (2006) 45:7384–7388.[CrossRef]
- Beissenhirtz MK, Elnathan R, Weizmann Y, Willner I. The aggregation of Au nanoparticles by an autonomous DNA machine detects viruses. Small (2007) 3:375–379.[CrossRef][Web of Science][Medline]
- Dirks RM, Pierce NA. Triggered amplification by hybridization chain reaction. PNAS (2004) 101:15275–15278.[Abstract/Free Full Text]
- Beissenhirtz MK, Willner I. DNA-based machines. Org. Biomol. Chem. (2006) 4:3392–3401.[CrossRef][Web of Science][Medline]
- Shlyahovsky B, Li D, Weizmann Y, Nowarski R, Kotler M, Willner I. Spotlighting of cocaine by an autonomous aptamer-based machine. J. Am. Chem. Soc. (2007) 129:3814–3815.[CrossRef][Web of Science][Medline]
- Tyagi S, Kramer FR. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. (1996) 14:303–308.[CrossRef][Web of Science][Medline]
- Tyagi S, Bratu DP, Kramer FR. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. (1998) 16:49–53.[CrossRef][Web of Science][Medline]
- Tsourkas A, Behlke1 MA, Bao G. Structure–function relationships of shared-stem and conventional molecular beacons. Nucleic Acids Res. (2002) 30:4208–4215.[Abstract/Free Full Text]
- Jay AL, Heinz FC, Robert AO. Virology (1994) 3rd edn. NJ: Prentice-Hall, Inc. A Paramount Communications Company.
CiteULike 
Connotea 
Del.icio.us What's this?
TOP
ABSTRACT
INTRODUCTION