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DNA probe amplification methods
Journal of ViroIogical Methods, 35 (1991) 117-126 0 1991 Elsevier Science Publishers B.V. / All rights reserved / 0166-0934/91/$03.50
117
VIRMET 01253
Mini-Review
DNA probe amplification methods Larry G. Birkenmeyer and Isa K. Mushahwar Experimental
Biology Research,
Abbott Laboratories,
(Accepted
North Chicago, Illinois. U.S.A.
19 July 1991)
Amplification method; Polymerase chain reaction; Ligase chain reaction; Nucleic acid sequence-based amplification; Ampliprobe; Q-beta replicase
Introduction
Nucleic acid probes and related techniques have become essential analytical methods for virtually all aspects of biological research in experimental biology and medicine (Tenover, 1988). These techniques require the introduction of a specifically labelled DNA (or RNA) probe capable of hybridizing with a complementary (target) sequence directly in a clinical sample. They consist of four essential steps, which are: sample preparation and denaturation; addition of labelled probe and hybridization; removal of unbound probe, and the detection of bound probe (Budd and Czerski, 1989). The sensitivity of detection for bound probe or labelled hybrid duplexes depends on the method utilized for this purpose. The last five years witnessed the introduction of several elegant methods for the detection of nucleic acid targets in addition to traditional methodologies such as slot-blot, Southern blot and direct solution hybridization. These recent methods are termed ‘amplification methods’ because they exploit the replicative functions of nucleic acids to amplify probe or target sequences (Engteberg, 1991). The purpose of this Mini-Review is to describe and compare briefly these amplification techniques which include the polymerase chain reaction (PCR), the-ligase chain reaction (LCRTM), self-sustained sequence rephcation (3SR), the Ampliprobe system and Q-beta replicase (Qfi).
Correspondence to: I.K. Mushahwar, North Chicago, IL 60064, U.S.A.
Ph.D., Experimental
Biology Research, Abbott
Laboratories,
118
Discussion
The basic concepts of the 5 amplification techniques are discussed below. The comparative features of these methods are shown in Tables 1 and 2. A detailed list of gene-amplification products and their suppliers is available in a recent review by Lewis (1991). Polymerase
chain reaction
The PCR is an in vitro method where specific nucleic acid sequences can be amplified (Saiki et al., 1985; Mullis and Faloona, 1987). In its simplest form (Fig. l), double-stranded target DNA is heat-denatured in the presence of a molar excess of 2 oligonucleotide primers. The 2 primers flank the sequence to be amplified (< 100 to 10000 bases) and are complementary to opposite strands of the target. At a reduced temperature the primers hybridize to the target DNA in a sequence-specific manner and are extended toward one another by a thermostable DNA polymerase (Saiki et al., 1988). The primer extension products are dissociated from the target template by heating. Each TABLE 1 Comparative Method
features of five DNA probe amplification Amplification” Target
Relative sensitivityb
Thermocycling required
Potential uses
High
Yes
Exponential
High High
Yes No
Exponential Linear
High Low
No No
Target detection, cloning, sequencing Target detection Target detection, cloning, sequencing Target detection Target detection
Signal
PCR
Exponential
LCRTM 3SR
Exponential
Qj replicase Ampliprobe
methods
“A method is described as target amplification only if the amplification products contain a sequence not present in the initial probes. bHigh = capable of detecting < IO3 molecules. Low = capable of detecting 2 lo4 molecules. TABLE 2 Other important Method
PCR LCRTM 3SR Qfl replicase Ampliprobe “1 for blunt-end
features of five DNA probe amplification
No. of targetspecific probes required
No. of
2
1
methods
Reaction time (h)
Detection time (h)
Automated detection
Sensitivity to contamination
;
24 <2
1
I
<1
l-2 40.8 1-2 60.8
No
1or2” 3
1
1
>2
1
High High High High Low
enzymes required
LCR, 2 for double-gap
LCR; bIMx system (Abbott
Yesb No No No
Laboratories).
119
extension product, along with the original target, can serve as a tempiate in a second round of primer annealing and extension. Thus, after n rounds of denaturation, annealing and extension, the target Sequence theoretically can be amplified 2”-fold. Ligase chain reaction
Ligase chain reaction is a highly sensitive assay for the detection of specific
3’ A
+,
B
5’
+ t A
A
r
B
+
0
I
~polymarase
Fig. 1. FCR amplification. Schematic representation of the effect of heating and cooling, in the presence of primers A and B (a) and a thermostable DNA polymerase on the amp~i~~tion of the target seqitence. Repeated cycling of the reaction is indicated by the brackets.
120
nucleic acid sequences in a sample. It is an amplification system which results in the exponential accumulation of reaction product over the course of the assay. In contrast to PCR, which requires 2 oligonucleotide primers, LCR utilizes 4 oligonucleotides to achieve its high level of sensitivity and specificity. In standard LCR (Backman, 1987) as shown in Fig. 2, 2 LCR oligonucleotides hybridize adjacent to one another on each of the denatured target DNA strands, such that a nick is formed. The nick is sealed by a thermostable DNA ligase (Backman et al., 1988). Each ligated product, along
target
3’
_.
5’
.
3
;
4
1.
I s
3
+
Heat
3
1
Y
4 *
1
;
3
i: 2
4 i
s
3’
T 2
+ -
ONAligSe
i 4
Fig. 2. LCR amplification. A nick is formed by juxtaposed oligonucieotides annealed to target. The nick is sealed (0) by DNA ligase.
121
with the original target, can serve as template in subsequent rounds of denaturation, annealing and 1iFation. A modification of this procedure (Fig. 3), called gapped LCR (G-LCR M), differs from LCR in that a short gap is formed after annealing of the oligonucleotides to the template. The gap is filled
O&onucleoUdes5’
1
+
3
3’
2
I
4
1
.-.A
3
5 3
5’ 3
2
2
4
+
’
1
3
w
DNA4~aa DNAligaee 3'
5' 3
Jvl,
1
+
2n/\
5'
1
3'
2
5'
1
5
2
A.
4”
” +/VI
AA --
: 4
Fig. 3. G-LCR amplification. A gap of a few bases is formed upon annealing of adjacent oligonucleotides. The gap is filled (saw tooth line) and ligated (0) by a thermostable polymerase and ligase respectively. For both LCR and G-LCR, denaturation and annealing are brought about by varying the reaction temperature. The brackets indicate repeated the~~ycling of the reaction.
122
DNA Target 5’
3
DNA DNA
5 = 3
RNA Target
1
5’
DNA DNA
I anneal
RNA DNA
5
t
3 n
I
RT
DNA DNA
RNA
RNA
., .,\,.,b,
Fig. 4. 3SR amplification. The sequential series of events that make up the 3SR reaction are shown for both RNA and DNA targets, either of which can be copied by reverse transcriptase (RT) when annealed with an appropriate primer. Single-stranded primers A and B are present in molar excess and are comprised of a target-specific region (-) and a region encoding the T7 RNA polymerase promoter (m). An angled box indicates that the promoter region is single-stranded and therefore inactive. A doublestranded promoter region to which T7 RNA polymerase (RNA pol) can bind and initiate transcription is shown as a horizontal box. The ability of RNase H to digest the RNA strand in a DNA:RNA hybrid is represented by the broken lines.
in by a thermostable DNA (Backman et al., 1991).
polymerase
and
the resulting
nick is ligated
123
Nucleic acid sequence-based amp&ation replication (3SR)
(also known as self-sustained sequence
Self-sustained sequence replication resembles PCR in that it utilizes 2 primers to exponentially amplify the sequence flanked by the primers (Guatelli et al., 1990). Unlike PCR, 3 enzymes are required and the reaction is performed at a constant temperature. The action of all 3 enzymes produces alternating waves of reverse transcription and transcription that result in a self-sustained amplification of the target sequence. The rather complex cascade (Fig. 4) of events that occur in 3SR is a modification from Guatelli et al. (1990). Ampliprobe
The Ampliprobe system (ImClone Systems, New York, NY, U.S.A.) as shown in Fig. 5, relies on amplification of’the detection signal rather than amplifying the target DNA itself. DNA, complementary to the desired target, is cloned into an Ml3 phage vector. Single-stranded DNA is isolated and hybridized against target DNA fixed to a solid support. Secondary probes, complementary to M13, bind to the vector sequence and are detected by alkaline phosphatase (AP). The nature of AP binding to the secondary probe is unclear, although possibilities include AP-labelled oligomers complementary to the secondary probe or binding of an AP-avidin conjugate to a biotinylated secondary probe. Q-beta replicase
Q-beta replicase catalyzes the self-replication of a 221-base RNA template designated MDV-I (Kacian et al., 1972). Over the course of 30 min, at a constant temperature, the replicase can boost the level of MDV-1 template a
AP
-
Ml 3 vector
Fig. 5. Ampliprobe system. An array of secondary probes, which can be detected through a reporter molecule such as alkaline phosphatase (AP), is shown complexed with target. The primary Ml3 probe contains sequences complementary to both the target and to the secondary probes, and so acts as a bridge between them.
124
billion fold. The MDV-1 RNA can be used solely as an amplifiable reporter group (Chu et al., 1986) as shown in Fig. 6a. For example, a biotinylated target-specific probe could be detected by adding avidin-linked MDV-1 RNA, removing unbound MDV-1 and then adding Q/I replicase. The resulting amplified MDV-1 copies can be detected by ethidium bromide staining or by a secondary probe homologous to MDV- 1. Alternatively, MDV-1 RNA can serve as both target-specific probe and detection signal amplifier (Lizardi et al., 1988) as shown in Fig. 6b. A targetspecific sequence is inserted into MDV-1, generating a hybrid RNA which is capable of hybridizing to the target. Non-hybridized probe is removed and the bound probe is amplified by the addition of Qfl replicase. Detection of the amplified hybrid RNAs can be performed as described above. Diagnostic applications Since the introduction of PCR, a variety of related assays have been developed and used in most fields of laboratory medicine. Many recent reviews have appeared in the literature concerning PCR technology and its application
MDV-1 RNA
a)
Probe
#
Target
Probe Insert + Cg3Replicase
Fig. 6. Q/3 replicase. (a) A probe annealed to target is linked (.) to MDV-1 RNA which, in the presence of Qj3 replicase, serves as a signal amplification system. (b) Proposed system in which MDV-I RNA contains a target-specific insert.
125
in diagnostic immunology (Erlich, 1989; Brechot, 1990; Eisenstein, 1990; Laure et al., 1990; Mullis, 1990; Remick et al., 1990). Articles: covering.the other 4 amplification methods have been published and are of interest. These include discussions on LCR (Wu and Wallace, 1989; Barany, 1991; Engleberg, 1991), Qfl (Lizardi et al., 1988; Lizardi and Kramer, 1991), and 3SR (Guatelli et al., 1990). It seems likely that these methods will continue to expand our capabilities to rapidly diagnose infectious diseases, cancer and genetic disorders. Future applications will also include areas such as veterinary medicine and forensic science (Compton, 1991). The current variety of amplification techniques and their applications will contribute importantly to discovery research and in practical ways to clinical medicine. References Backman, K. (1987) Method for detecting a target nucleic acid sequence. European Patent Office 320,308. Priority, December 11, 1987. Backman, K., Rudd, L., Lauer, G. and McKay, D. (1988) Isolating thermostable enzymes. European Patent Office - 373,962. Priority, December 16, 1988. Backman, K., Carrino, J.J., Bond, S.B. and Laffler, T.G. (1991) Improved method of amplifying target nucleic acids applicable to both polymerase and hgase chain reactions. European Patent Office - 439, 182A. Priority January 26, 1990. Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Nat]. Acad. Sci. USA 88, 189-193. B&hot, C. (1990) Polymerase chain reaction, a new tool for the study of viral infections in hepatology. J. Hepatol. 11, 124129. Budd, R.A. and Czerski, P. (1988) Applications of DNA probes for the diagnosis of human infectious diseases: an overview. U.S. Department of Health and Human Services, Public Health Service, Food and Drug Administration Publication No. (FDA) 88-4229, pp. I-30. Chu, B.C.F., Kramer, F.R. and Orgel, L.E. (1986) Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res. 14, 5591-5603. Compton, J. (1991) Nucleic acid sequence-based amplification. Nature 350, 91-92. Eisenstein, B.I. (1990) The polymerase chain reaction: a new method using molecular genetics for medical diagnosis. New Engl. J. Med. 322, 178-183. Engleberg, N.C. (1991) Nucleic acid probe tests for clinical diagnosis-where do we stand? Am. Sot. Microbial. News 57, 183-186. Erlich, H.A. (1989) Polymerase chain reaction. J. Clin. Immunol. 9, 43747. Guatelli, J.C., Whittield, K.M., Kwob, D.Y., Barringer, K.J., Richman, D.D. and Gingeras, T.R. (1990) Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87, 18741878. Kacian, D.L., Mills, D.R., Kramer, F.R. and Spiegelman, S. (1972) A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication. Proc. Natl. Acad. Sci. USA 69, 3038-3042. Laure, F., Courgnaud, V. and B&hot, C. (1990) The polymerase chain reaction in the human immunodeficiency virus diagnosis. Ann. Biol. Clin. 48, 413415. Lewis, R. (1991) Innovative alternatives to PCR technology are proliferating. The Scientist (January 21, 1991), 23-24. Lizardi, P.M., Guerra, C.E., Lomeli, H., Tussie-Luna, I. and Kramer, F.R. (1988) Exponential amplification of recombinant-RNA hybridization probes. BioTechnology 6, 1197-1202. Lizardi, P.M. and Kramer, F.R. (1991) Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases. Trends Biotechnol. 9, 53-58.
126 Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Sci. Am. (April 1990) 56 65. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol. 155, 335-350. Remick, D.G., Kunkel, S.L., Holbrook, E.A. and Hanson, C.A. (1990) Theory and applications of the polymerase chain reaction. Am. J. Clin. Pathol. 93, S49-S54. Saiki, R.K., Scharf, S., Faloona, F.A., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of /?-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. Tenover, F.C. (1988) Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbial. Rev. I. 82-101. Wu, D.Y. and Wallace, R.B. (1989) The ligation amplification reaction (LAR) - amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics 4, 560-569.
117
VIRMET 01253
Mini-Review
DNA probe amplification methods Larry G. Birkenmeyer and Isa K. Mushahwar Experimental
Biology Research,
Abbott Laboratories,
(Accepted
North Chicago, Illinois. U.S.A.
19 July 1991)
Amplification method; Polymerase chain reaction; Ligase chain reaction; Nucleic acid sequence-based amplification; Ampliprobe; Q-beta replicase
Introduction
Nucleic acid probes and related techniques have become essential analytical methods for virtually all aspects of biological research in experimental biology and medicine (Tenover, 1988). These techniques require the introduction of a specifically labelled DNA (or RNA) probe capable of hybridizing with a complementary (target) sequence directly in a clinical sample. They consist of four essential steps, which are: sample preparation and denaturation; addition of labelled probe and hybridization; removal of unbound probe, and the detection of bound probe (Budd and Czerski, 1989). The sensitivity of detection for bound probe or labelled hybrid duplexes depends on the method utilized for this purpose. The last five years witnessed the introduction of several elegant methods for the detection of nucleic acid targets in addition to traditional methodologies such as slot-blot, Southern blot and direct solution hybridization. These recent methods are termed ‘amplification methods’ because they exploit the replicative functions of nucleic acids to amplify probe or target sequences (Engteberg, 1991). The purpose of this Mini-Review is to describe and compare briefly these amplification techniques which include the polymerase chain reaction (PCR), the-ligase chain reaction (LCRTM), self-sustained sequence rephcation (3SR), the Ampliprobe system and Q-beta replicase (Qfi).
Correspondence to: I.K. Mushahwar, North Chicago, IL 60064, U.S.A.
Ph.D., Experimental
Biology Research, Abbott
Laboratories,
118
Discussion
The basic concepts of the 5 amplification techniques are discussed below. The comparative features of these methods are shown in Tables 1 and 2. A detailed list of gene-amplification products and their suppliers is available in a recent review by Lewis (1991). Polymerase
chain reaction
The PCR is an in vitro method where specific nucleic acid sequences can be amplified (Saiki et al., 1985; Mullis and Faloona, 1987). In its simplest form (Fig. l), double-stranded target DNA is heat-denatured in the presence of a molar excess of 2 oligonucleotide primers. The 2 primers flank the sequence to be amplified (< 100 to 10000 bases) and are complementary to opposite strands of the target. At a reduced temperature the primers hybridize to the target DNA in a sequence-specific manner and are extended toward one another by a thermostable DNA polymerase (Saiki et al., 1988). The primer extension products are dissociated from the target template by heating. Each TABLE 1 Comparative Method
features of five DNA probe amplification Amplification” Target
Relative sensitivityb
Thermocycling required
Potential uses
High
Yes
Exponential
High High
Yes No
Exponential Linear
High Low
No No
Target detection, cloning, sequencing Target detection Target detection, cloning, sequencing Target detection Target detection
Signal
PCR
Exponential
LCRTM 3SR
Exponential
Qj replicase Ampliprobe
methods
“A method is described as target amplification only if the amplification products contain a sequence not present in the initial probes. bHigh = capable of detecting < IO3 molecules. Low = capable of detecting 2 lo4 molecules. TABLE 2 Other important Method
PCR LCRTM 3SR Qfl replicase Ampliprobe “1 for blunt-end
features of five DNA probe amplification
No. of targetspecific probes required
No. of
2
1
methods
Reaction time (h)
Detection time (h)
Automated detection
Sensitivity to contamination
;
24 <2
1
I
<1
l-2 40.8 1-2 60.8
No
1or2” 3
1
1
>2
1
High High High High Low
enzymes required
LCR, 2 for double-gap
LCR; bIMx system (Abbott
Yesb No No No
Laboratories).
119
extension product, along with the original target, can serve as a tempiate in a second round of primer annealing and extension. Thus, after n rounds of denaturation, annealing and extension, the target Sequence theoretically can be amplified 2”-fold. Ligase chain reaction
Ligase chain reaction is a highly sensitive assay for the detection of specific
3’ A
+,
B
5’
+ t A
A
r
B
+
0
I
~polymarase
Fig. 1. FCR amplification. Schematic representation of the effect of heating and cooling, in the presence of primers A and B (a) and a thermostable DNA polymerase on the amp~i~~tion of the target seqitence. Repeated cycling of the reaction is indicated by the brackets.
120
nucleic acid sequences in a sample. It is an amplification system which results in the exponential accumulation of reaction product over the course of the assay. In contrast to PCR, which requires 2 oligonucleotide primers, LCR utilizes 4 oligonucleotides to achieve its high level of sensitivity and specificity. In standard LCR (Backman, 1987) as shown in Fig. 2, 2 LCR oligonucleotides hybridize adjacent to one another on each of the denatured target DNA strands, such that a nick is formed. The nick is sealed by a thermostable DNA ligase (Backman et al., 1988). Each ligated product, along
target
3’
_.
5’
.
3
;
4
1.
I s
3
+
Heat
3
1
Y
4 *
1
;
3
i: 2
4 i
s
3’
T 2
+ -
ONAligSe
i 4
Fig. 2. LCR amplification. A nick is formed by juxtaposed oligonucieotides annealed to target. The nick is sealed (0) by DNA ligase.
121
with the original target, can serve as template in subsequent rounds of denaturation, annealing and 1iFation. A modification of this procedure (Fig. 3), called gapped LCR (G-LCR M), differs from LCR in that a short gap is formed after annealing of the oligonucleotides to the template. The gap is filled
O&onucleoUdes5’
1
+
3
3’
2
I
4
1
.-.A
3
5 3
5’ 3
2
2
4
+
’
1
3
w
DNA4~aa DNAligaee 3'
5' 3
Jvl,
1
+
2n/\
5'
1
3'
2
5'
1
5
2
A.
4”
” +/VI
AA --
: 4
Fig. 3. G-LCR amplification. A gap of a few bases is formed upon annealing of adjacent oligonucleotides. The gap is filled (saw tooth line) and ligated (0) by a thermostable polymerase and ligase respectively. For both LCR and G-LCR, denaturation and annealing are brought about by varying the reaction temperature. The brackets indicate repeated the~~ycling of the reaction.
122
DNA Target 5’
3
DNA DNA
5 = 3
RNA Target
1
5’
DNA DNA
I anneal
RNA DNA
5
t
3 n
I
RT
DNA DNA
RNA
RNA
., .,\,.,b,
Fig. 4. 3SR amplification. The sequential series of events that make up the 3SR reaction are shown for both RNA and DNA targets, either of which can be copied by reverse transcriptase (RT) when annealed with an appropriate primer. Single-stranded primers A and B are present in molar excess and are comprised of a target-specific region (-) and a region encoding the T7 RNA polymerase promoter (m). An angled box indicates that the promoter region is single-stranded and therefore inactive. A doublestranded promoter region to which T7 RNA polymerase (RNA pol) can bind and initiate transcription is shown as a horizontal box. The ability of RNase H to digest the RNA strand in a DNA:RNA hybrid is represented by the broken lines.
in by a thermostable DNA (Backman et al., 1991).
polymerase
and
the resulting
nick is ligated
123
Nucleic acid sequence-based amp&ation replication (3SR)
(also known as self-sustained sequence
Self-sustained sequence replication resembles PCR in that it utilizes 2 primers to exponentially amplify the sequence flanked by the primers (Guatelli et al., 1990). Unlike PCR, 3 enzymes are required and the reaction is performed at a constant temperature. The action of all 3 enzymes produces alternating waves of reverse transcription and transcription that result in a self-sustained amplification of the target sequence. The rather complex cascade (Fig. 4) of events that occur in 3SR is a modification from Guatelli et al. (1990). Ampliprobe
The Ampliprobe system (ImClone Systems, New York, NY, U.S.A.) as shown in Fig. 5, relies on amplification of’the detection signal rather than amplifying the target DNA itself. DNA, complementary to the desired target, is cloned into an Ml3 phage vector. Single-stranded DNA is isolated and hybridized against target DNA fixed to a solid support. Secondary probes, complementary to M13, bind to the vector sequence and are detected by alkaline phosphatase (AP). The nature of AP binding to the secondary probe is unclear, although possibilities include AP-labelled oligomers complementary to the secondary probe or binding of an AP-avidin conjugate to a biotinylated secondary probe. Q-beta replicase
Q-beta replicase catalyzes the self-replication of a 221-base RNA template designated MDV-I (Kacian et al., 1972). Over the course of 30 min, at a constant temperature, the replicase can boost the level of MDV-1 template a
AP
-
Ml 3 vector
Fig. 5. Ampliprobe system. An array of secondary probes, which can be detected through a reporter molecule such as alkaline phosphatase (AP), is shown complexed with target. The primary Ml3 probe contains sequences complementary to both the target and to the secondary probes, and so acts as a bridge between them.
124
billion fold. The MDV-1 RNA can be used solely as an amplifiable reporter group (Chu et al., 1986) as shown in Fig. 6a. For example, a biotinylated target-specific probe could be detected by adding avidin-linked MDV-1 RNA, removing unbound MDV-1 and then adding Q/I replicase. The resulting amplified MDV-1 copies can be detected by ethidium bromide staining or by a secondary probe homologous to MDV- 1. Alternatively, MDV-1 RNA can serve as both target-specific probe and detection signal amplifier (Lizardi et al., 1988) as shown in Fig. 6b. A targetspecific sequence is inserted into MDV-1, generating a hybrid RNA which is capable of hybridizing to the target. Non-hybridized probe is removed and the bound probe is amplified by the addition of Qfl replicase. Detection of the amplified hybrid RNAs can be performed as described above. Diagnostic applications Since the introduction of PCR, a variety of related assays have been developed and used in most fields of laboratory medicine. Many recent reviews have appeared in the literature concerning PCR technology and its application
MDV-1 RNA
a)
Probe
#
Target
Probe Insert + Cg3Replicase
Fig. 6. Q/3 replicase. (a) A probe annealed to target is linked (.) to MDV-1 RNA which, in the presence of Qj3 replicase, serves as a signal amplification system. (b) Proposed system in which MDV-I RNA contains a target-specific insert.
125
in diagnostic immunology (Erlich, 1989; Brechot, 1990; Eisenstein, 1990; Laure et al., 1990; Mullis, 1990; Remick et al., 1990). Articles: covering.the other 4 amplification methods have been published and are of interest. These include discussions on LCR (Wu and Wallace, 1989; Barany, 1991; Engleberg, 1991), Qfl (Lizardi et al., 1988; Lizardi and Kramer, 1991), and 3SR (Guatelli et al., 1990). It seems likely that these methods will continue to expand our capabilities to rapidly diagnose infectious diseases, cancer and genetic disorders. Future applications will also include areas such as veterinary medicine and forensic science (Compton, 1991). The current variety of amplification techniques and their applications will contribute importantly to discovery research and in practical ways to clinical medicine. References Backman, K. (1987) Method for detecting a target nucleic acid sequence. European Patent Office 320,308. Priority, December 11, 1987. Backman, K., Rudd, L., Lauer, G. and McKay, D. (1988) Isolating thermostable enzymes. European Patent Office - 373,962. Priority, December 16, 1988. Backman, K., Carrino, J.J., Bond, S.B. and Laffler, T.G. (1991) Improved method of amplifying target nucleic acids applicable to both polymerase and hgase chain reactions. European Patent Office - 439, 182A. Priority January 26, 1990. Barany, F. (1991) Genetic disease detection and DNA amplification using cloned thermostable ligase. Proc. Nat]. Acad. Sci. USA 88, 189-193. B&hot, C. (1990) Polymerase chain reaction, a new tool for the study of viral infections in hepatology. J. Hepatol. 11, 124129. Budd, R.A. and Czerski, P. (1988) Applications of DNA probes for the diagnosis of human infectious diseases: an overview. U.S. Department of Health and Human Services, Public Health Service, Food and Drug Administration Publication No. (FDA) 88-4229, pp. I-30. Chu, B.C.F., Kramer, F.R. and Orgel, L.E. (1986) Synthesis of an amplifiable reporter RNA for bioassays. Nucleic Acids Res. 14, 5591-5603. Compton, J. (1991) Nucleic acid sequence-based amplification. Nature 350, 91-92. Eisenstein, B.I. (1990) The polymerase chain reaction: a new method using molecular genetics for medical diagnosis. New Engl. J. Med. 322, 178-183. Engleberg, N.C. (1991) Nucleic acid probe tests for clinical diagnosis-where do we stand? Am. Sot. Microbial. News 57, 183-186. Erlich, H.A. (1989) Polymerase chain reaction. J. Clin. Immunol. 9, 43747. Guatelli, J.C., Whittield, K.M., Kwob, D.Y., Barringer, K.J., Richman, D.D. and Gingeras, T.R. (1990) Isothermal, in vitro amplification of nucleic acids by a multienzyme reaction modeled after retroviral replication. Proc. Natl. Acad. Sci. USA 87, 18741878. Kacian, D.L., Mills, D.R., Kramer, F.R. and Spiegelman, S. (1972) A replicating RNA molecule suitable for a detailed analysis of extracellular evolution and replication. Proc. Natl. Acad. Sci. USA 69, 3038-3042. Laure, F., Courgnaud, V. and B&hot, C. (1990) The polymerase chain reaction in the human immunodeficiency virus diagnosis. Ann. Biol. Clin. 48, 413415. Lewis, R. (1991) Innovative alternatives to PCR technology are proliferating. The Scientist (January 21, 1991), 23-24. Lizardi, P.M., Guerra, C.E., Lomeli, H., Tussie-Luna, I. and Kramer, F.R. (1988) Exponential amplification of recombinant-RNA hybridization probes. BioTechnology 6, 1197-1202. Lizardi, P.M. and Kramer, F.R. (1991) Exponential amplification of nucleic acids: new diagnostics using DNA polymerases and RNA replicases. Trends Biotechnol. 9, 53-58.
126 Mullis, K.B. (1990) The unusual origin of the polymerase chain reaction. Sci. Am. (April 1990) 56 65. Mullis, K.B. and Faloona, F.A. (1987) Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol. 155, 335-350. Remick, D.G., Kunkel, S.L., Holbrook, E.A. and Hanson, C.A. (1990) Theory and applications of the polymerase chain reaction. Am. J. Clin. Pathol. 93, S49-S54. Saiki, R.K., Scharf, S., Faloona, F.A., Mullis, K.B., Horn, G.T., Erlich, H.A. and Arnheim, N. (1985) Enzymatic amplification of /?-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350-1354. Tenover, F.C. (1988) Diagnostic deoxyribonucleic acid probes for infectious diseases. Clin. Microbial. Rev. I. 82-101. Wu, D.Y. and Wallace, R.B. (1989) The ligation amplification reaction (LAR) - amplification of specific DNA sequences using sequential rounds of template-dependent ligation. Genomics 4, 560-569.