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1.
Anal Biochem ; 276(2): 177-87, 1999 Dec 15.
Article in English | MEDLINE | ID: mdl-10603241

ABSTRACT

Strand displacement amplification (SDA) is an isothermal nucleic acid amplification method based on the primer-directed nicking activity of a restriction enzyme and the strand displacement activity of an exonuclease-deficient polymerase. Here we describe fluorogenic reporter probes that permit real-time, sequence-specific detection of targets amplified during SDA. The new probes possess the single-strand half of a BsoBI recognition sequence flanked on opposite sides by a fluorophore and a quencher. The probes also contain target-binding sequences located 3' to the BsoBI site. Fluorophore and quencher are maintained in sufficiently close proximity that fluorescence is quenched in the intact single-stranded probe. If target is present during SDA, the probe is converted into a fully double-stranded form and is cleaved by the restriction enzyme BsoBI, which also serves as the nicking agent for SDA. Fluorophore and quencher diffuse apart upon probe cleavage, causing increased fluorescence. Target replication may thus be followed in real time during the SDA reaction. Probe performance may be enhanced by embedding the fluorogenic BsoBI site within the loop of a folded hairpin structure. The new probe designs permit detection of as few as 10 target copies within 30 min in a closed-tube, real-time format, eliminating the possibility of carry-over contamination. The probes may be used to detect RNA targets in SDA mixtures containing reverse transcriptase. Furthermore, a two-color competitive SDA format permits accurate quantification of target levels from the real-time fluorescence data.


Subject(s)
Nucleic Acid Amplification Techniques , Nucleic Acids/analysis , Base Sequence , Chlamydia/genetics , DNA, Bacterial/genetics , DNA, Viral/genetics , Fluorescent Dyes , Genes, gag , HIV/genetics , Molecular Probe Techniques , Nucleic Acids/genetics , Oligonucleotide Probes/genetics , RNA, Bacterial/analysis , RNA, Bacterial/genetics , RNA, Viral/analysis , RNA, Viral/genetics
2.
Anal Biochem ; 247(1): 130-7, 1997 Apr 05.
Article in English | MEDLINE | ID: mdl-9126382

ABSTRACT

Strand displacement amplification (SDA) is an isothermal DNA amplification technology that uses a restriction enzyme and polymerase. We have developed a target-specific method which allows simultaneous SDA and detection in a homogeneous format. This is accomplished by including a detector oligodeoxynucleotide labeled with 5-(4,6-dichlorotriazin-2-yl)amino fluorescein in the SDA reaction. Fluorescence polarization is used to monitor hybridization of the detector probe to the amplification product as it rises in concentration during SDA. We have demonstrated real-time SDA detection for the cryptic plasmid of Chlamydia trachomatis with high sensitivity in only 30 min.


Subject(s)
Chlamydia trachomatis/genetics , Chlamydia trachomatis/isolation & purification , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Fluorescence Polarization/methods , Nucleic Acid Amplification Techniques , Base Sequence , Chlamydia Infections/diagnosis , Evaluation Studies as Topic , Female , Fluorescein , Fluoresceins , Fluorescence Polarization/statistics & numerical data , Fluorescent Dyes , Humans , Male , Oligonucleotide Probes/genetics , Sensitivity and Specificity , Time Factors
3.
Anal Chem ; 69(3): 500-6, 1997 Feb 01.
Article in English | MEDLINE | ID: mdl-9030058

ABSTRACT

The effects of temperature and collisional quenching on fluorescence polarization detection of DNA hybridization were studied using measurements of fluorescence intensity and anisotropy and the dynamic decay of these properties. Three different tethers, 3, 6, and 12 carbons in length, were used to attach fluorescein label to the 5' end of the 33-base oligomers. Perrin plots showed that the effective rotating volume decreases with increasing tether length and approximately doubles upon hybridization. Hybridization increases the association between the tethered dye and the DNA for the shorter tethers but displaces the fluorescein on the 12C tether from the DNA, forcing it into greater contact with the bulk solution. The 6C tether appears to promote sequence-specific interaction between fluorescein label and the oligomer, which causes unexpectedly high anisotropy at higher temperatures and increased protection from collisional quenching. In all cases, there appear to exist several possible conformations for the tethered fluorescein. As temperature is increased, these conformations tend to collapse into a single, average or preferred, conformation. The results demonstrate the importance of the selection of tether, dye, and DNA probe in designing a polarization strategy for detection of DNA hybridization, particularly with respect to tether length and DNA probe sequence.


Subject(s)
DNA/analysis , Nucleic Acid Hybridization , Anisotropy , Fluorescence Polarization , Thermodynamics
4.
Clin Chem ; 42(10): 1604-8, 1996 Oct.
Article in English | MEDLINE | ID: mdl-8855142

ABSTRACT

Strand displacement amplification (SDA) is an isothermal, in vitro method for diagnostics that amplifies a target DNA sequence by using a restriction enzyme and DNA polymerase. We have combined a new thermophilic form of SDA that involves restriction enzyme BsoBI and polymerase exo-Bca with fluorescence polarization for detection of Mycobacterium tuberculosis DNA by using the IS6110 insertion element as the target sequence. A 5'-fluorescein-labeled oligodeoxynucleotide detector probe hybridizes to the amplified product as it rises in concentration during SDA, and the single- to double-stranded conversion is monitored through an increase in fluorescence polarization. The associated change in polarization upon amplification of the target sequence is enhanced by specific polymerase binding to the double-stranded detector probe. Fewer than 10 M. tuberculosis genomes can be amplified and detected with an extremely simple protocol that takes only 20 min and uses relatively simple instrumentation and reagents, all of which can be purchased off-the-shelf.


Subject(s)
DNA, Bacterial/analysis , Fluorescence Polarization , Mycobacterium tuberculosis/genetics , Nucleic Acid Amplification Techniques , DNA Primers , DNA Restriction Enzymes , DNA-Directed DNA Polymerase , Deoxyribonuclease EcoRI , Fluorescein , Fluoresceins , Nucleic Acid Hybridization , Oligonucleotide Probes , Sensitivity and Specificity
5.
Nucleic Acids Res ; 24(2): 348-53, 1996 Jan 15.
Article in English | MEDLINE | ID: mdl-8628661

ABSTRACT

Strand displacement amplification (9SDA) is an isothermal in vitro method of amplifying a DNA sequence prior to its detection. We have combined SDA with fluorescence polarization detection. A 5'-fluorescein-labelled oligodeoxynucleotide detector probe hybridizes to the amplification product that rises in concentration during SDA and the single- to double strand conversion is monitored through an increase in fluorescence polarization. Detection sensitivity can be enhanced by using a detector probe containing an EcoRI recognition sequence at its 5'-end that is not homologous to the target sequence. During SDA the probe is converted to a fully double-stranded form that specifically binds a genetically modified form of the endonuclease EcoRI which lacks cleavage activity but retains binding specificity. We have applied this SDA detection system to a target sequence specific for Mycobacterium tuberculosis.


Subject(s)
DNA-Binding Proteins , DNA/analysis , Escherichia coli Proteins , Fluorescence Polarization/methods , Nucleic Acid Amplification Techniques , Base Sequence , DNA, Bacterial/analysis , Deoxyribonuclease EcoRI , Fluorescein , Fluoresceins , Molecular Probes , Molecular Sequence Data , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification
6.
Clin Chem ; 42(1): 9-13, 1996 Jan.
Article in English | MEDLINE | ID: mdl-8565240

ABSTRACT

Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a DNA sequence for diagnostic purposes. We have combined SDA with fluorescence polarization detection in a closed, homogeneous format. A fluorescently labeled oligodeoxynucleotide detector probe hybridizes to the amplification product that increases in concentration during SDA. The single- to double-stranded conversion of the probe is accompanied by an increase in fluorescence polarization values, which can be measured in real-time without physical manipulation of the sample. The probe was labeled with the near-infrared dye La Jolla Blue, and fluorescence polarization was measured on a transient-state fluorometer. We have applied this homogeneous SDA/detection system to a target DNA sequence specific for Mycobacterium tuberculosis DNA.


Subject(s)
DNA, Bacterial/analysis , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Amplification Techniques , Base Sequence , DNA Primers , DNA Probes , False Positive Reactions , Fluorescence Polarization , Molecular Sequence Data , Mycobacterium tuberculosis/genetics
7.
Mol Cell Probes ; 9(6): 399-403, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8808310

ABSTRACT

Strand displacement amplification (SDA) is an isothermal, in vitro method of amplifying a target DNA sequence. We performed SDA in the presence of a 5'-32P-oligodeoxynucleotide detector probe that contains a target binding sequence at its 3'-end and a recognition site for the restriction enzyme HincII at its 5'-end which is not homologous to the target sequence. The single-stranded probe hybridizes to the rising concentration of amplified product during SDA and is converted to a fully double-stranded form that is cleaved by HincII, releasing a 32P-labelled 5-mer fragment. Uncleaved probe (42-mer) and cleaved probe (5-mer) were separated by either gel electrophoresis or size exclusion filtration using a commercially available microcentrifuge device. The combined SDA/filtration protocol is simple and provides detection of as few as 10 molecules of target DNA. We applied the technique to detection of M. tuberculosis DNA.


Subject(s)
DNA Probes/analysis , Nucleic Acid Amplification Techniques , Base Sequence , Centrifugation , DNA/genetics , DNA/metabolism , DNA Polymerase I , DNA Probes/isolation & purification , DNA Probes/metabolism , DNA, Bacterial/analysis , DNA, Bacterial/genetics , DNA, Bacterial/isolation & purification , DNA, Single-Stranded/genetics , Deoxyribonucleases, Type II Site-Specific , Electrophoresis, Polyacrylamide Gel , Microchemistry , Molecular Sequence Data , Mycobacterium tuberculosis/genetics , Mycobacterium tuberculosis/isolation & purification , Nucleic Acid Denaturation , Phosphorus Radioisotopes/analysis , Ultrafiltration
8.
Anal Chem ; 67(21): 3945-51, 1995 Nov 01.
Article in English | MEDLINE | ID: mdl-8633758

ABSTRACT

Fundamental aspects of the application of fluorescence anisotropy to detect the hybridization of fluorescein-labeled DNA oligomers were explored. The oligomers included a binding site for the EcoRI restriction enzyme, which binds to double-stranded DNA and is used in this work to enhance the difference between the anisotropies of the single-stranded and double-stranded oligomers by increasing the effective volume of the latter. The fluorescence anisotropy increases upon hybridization and further upon binding of EcoRI to the double strand. By varying the length of the tether used to attach the fluorescein to the 5' end of the oligonucleotide, it was found that a 6-carbon tether was optimal, providing the most dramatic increases in anisotropy in the presence of EcoRI. Dynamic fluorescence anisotropy (DFA) provided insight into the increases in steady-state anisotropy. In most cases, the best fits to the DFA data were obtained using a biexponential decay model, which describes an anisotropic rotator. Upon hybridization, the faster rotational motion is more hindered, and the contribution of the slower rotational component is increased. This effect is enhanced by binding of EcoRI to the double strand, especially when the EcoRI binding site is near the fluorescein at the 5' end and the tether length is in the optimal range. Because the rotational correlation time of the slower anisotropy decay component is much longer than the fluorescence lifetime, it is possible in some cases to reduce the anisotropic rotator model to the special limiting case of a hindered rotator.


Subject(s)
DNA Probes/metabolism , DNA, Single-Stranded/chemistry , DNA/chemistry , Fluoresceins/chemistry , Fluorescence Polarization , Base Sequence , Binding Sites , DNA/metabolism , DNA, Single-Stranded/metabolism , Deoxyribonuclease EcoRI/metabolism , Fluorescein , Molecular Sequence Data , Nucleic Acid Hybridization , Protein Binding
10.
J Neurosci ; 12(6): 2156-64, 1992 Jun.
Article in English | MEDLINE | ID: mdl-1607934

ABSTRACT

[Ca2+]i was measured using fura-2-loaded isolated catfish horizontal cells in the presence of L-glutamate and the glutamate analogs kainate (KA), quisqualate (QA), and NMDA. Caffeine was used to release Ca2+ from intracellular stores. Cell membrane potential was controlled with a voltage clamp to prevent activation of voltage-dependent Ca2+ channels in the presence of agonist. All excitatory amino acid agonists produced a rapid and sustained rise in [Ca2+]i with the order of potency being QA greater than Glu greater than KA greater than NMDA. The agonist-induced [Ca2+]i increase was blocked in reduced [Ca2+]o and by 6-cyano-7-nitroquinoxaline-2,3-dione and 2-amino-5-phosphonopentanoate, which are specific blockers for QA/KA and NMDA receptors, respectively. The metabotropic receptor agonist trans-1-amino-1,3-cyclopentanedicarboxylic acid (ACPD; 10-200 microM) had no effect on [Ca2+]i. Hill coefficients from curves fitted to concentration-response data suggested an amplification of the Ca2+ signal that was interpreted as calcium-induced calcium release (CICR) from intracellular Ca2+ stores. Caffeine (10 mM) produced a rapid transient rise in [Ca2+]i, confirming the existence of a Ca(2+)-sensitive store. Following caffeine-induced depletion of Ca2+ from intracellular stores, agonists were still able to produce increases in [Ca2+]i, confirming Ca2+ influx through the agonist-gated channel. The agonist-induced increase in [Ca2+]i was decreased following caffeine-induced depletion, confirming a process of CICR. These results are consistent with the hypothesis that excitatory amino acids can produce direct modulation of [Ca2+]i by influx through the agonist-gated channel and by CICR from intracellular stores.


Subject(s)
Amino Acids/physiology , Calcium/metabolism , Intracellular Membranes/metabolism , Photoreceptor Cells/metabolism , Animals , Caffeine/pharmacology , Cell Separation , Electrophysiology , Ictaluridae , Intracellular Membranes/physiology , Ion Channel Gating , Osmolar Concentration , Photoreceptor Cells/physiology
11.
Anal Biochem ; 200(2): 400-4, 1992 Feb 01.
Article in English | MEDLINE | ID: mdl-1301067

ABSTRACT

A new fluorogenic substrate for the pyridoxal 5'-phosphate-dependent enzyme tryptophanase is described. L-Serine, which is linked to 7-amino-4-methylcoumarin through an O-carbamoyl tether, serves as a substrate for the enzyme. The released moiety, 7-amino-4-methylcoumarin (AMC), can be detected by either absorbance (355 nm) or fluorescence (excitation 365 nm/emission 440 nm). Kinetic constants were measured using each of these techniques: Km = 85 +/- 20 microM, Vmax = 2.9 +/- 0.4 mumol/min/mg (fluorescence) and Km = 129 +/- 21 microM, Vmax = 3.1 +/- 0.3 mumol/min/mg (absorbance). The Vmax for serine-AMC-carbamate is approximately 1.9 times faster than that of the natural substrate, tryptophan. Using fluorescence detection, solutions containing 10(-3) units of activity could be routinely assayed.


Subject(s)
Coumarins/analysis , Coumarins/chemical synthesis , Serine/analogs & derivatives , Tryptophanase/analysis , Coumarins/metabolism , Fluorescent Dyes , Indicators and Reagents , Kinetics , Molecular Structure , Serine/chemical synthesis , Serine/metabolism , Spectrometry, Fluorescence/methods , Substrate Specificity , Tryptophanase/metabolism
12.
Anal Biochem ; 179(2): 229-35, 1989 Jun.
Article in English | MEDLINE | ID: mdl-2774171

ABSTRACT

A method in which a two-enzyme cascade is used for rapid and sensitive detection of alkaline phosphatase is described. A masked inhibitor, 4-(3-oxo-4,4,4-trifluorobutyl)phenyl phosphate, is dephosphorylated by the action of alkaline phosphatase. The resulting compound, 1,1,1-trifluoro-4-(4-hydroxyphenyl)-butan-2-one, acts as a potent inhibitor of the second enzyme, a liver carboxylesterase. A determination of the residual esterase activity provides a highly sensitive indication of the original phosphatase concentration. The sensitivity of this dual-enzyme cascade is approximately 125-fold greater than that observed for the direct detection of phosphatase activity with p-nitrophenyl phosphate.


Subject(s)
Alkaline Phosphatase/analysis , Alkaline Phosphatase/antagonists & inhibitors , Chemical Phenomena , Chemistry , Esterases/metabolism
13.
J Med Chem ; 29(9): 1796-8, 1986 Sep.
Article in English | MEDLINE | ID: mdl-3746825

ABSTRACT

Three 4,5-disubstituted primaquine analogues were synthesized and evaluated for radical curative activity against Plasmodium cynomolgi in rhesus monkeys. One of the compounds showed moderate activity; however, none of the three compounds were as active as the lead compounds 5-methoxy-4-methylprimaquine and 4-(methoxy-methyl)-5-[m-(trifluoromethyl)phenoxy]primaquine.


Subject(s)
Malaria/drug therapy , Primaquine/analogs & derivatives , Animals , Chemical Phenomena , Chemistry , Macaca mulatta , Primaquine/therapeutic use , Structure-Activity Relationship
14.
J Med Chem ; 28(12): 1959-62, 1985 Dec.
Article in English | MEDLINE | ID: mdl-4068013

ABSTRACT

Two isomeric sulfur-interrupted 8-amino side chain analogues of 4-methyl-5-[m-(trifluoromethyl)phenoxy]primaquine (2) were prepared and tested for antimalarial activity. The compounds were evaluated for blood schizonticidal activity against Plasmodium berghei in mice and radical curative activity against Plasmodium cynomolgi in rhesus monkeys. In addition, they were evaluated for causal prophylactic activity against Plasmodium berghei yoelii in mice. Both compounds were more active and less toxic than primaquine in the P. berghei screen. One of the compounds showed radical curative activity similar to primaquine but was less active than 2. One of the compounds was active at 160 mg/kg in the P. berghei yoelii screen; the other was not active.


Subject(s)
Aminoquinolines/therapeutic use , Malaria/drug therapy , Aminoquinolines/chemical synthesis , Aminoquinolines/toxicity , Animals , Chemical Phenomena , Chemistry , Macaca mulatta , Malaria/prevention & control , Mice , Plasmodium , Primaquine/therapeutic use , Primaquine/toxicity
15.
J Med Chem ; 28(11): 1564-7, 1985 Nov.
Article in English | MEDLINE | ID: mdl-4067985

ABSTRACT

Five 4-substituted 5-[m-(trifluoromethyl)phenoxy]primaquine analogues were synthesized and tested for radical curative activity against Plasmodium cynomolgi in Rhesus monkeys and for blood schizonticidal antimalarial activity against Plasmodium berghei in mice. In addition, they were evaluated for causal prophylactic antimalarial activity against Plasmodium berghei yoelii in mice. One compound, 4-ethyl-5-[m-(trifluoromethyl)phenoxy]primaquine (2b), showed radical curative activity equivalent to 4-methyl-5-[m-(trifluoromethyl)phenoxy]primaquine (2a). A second compound showed radical curative activity slightly less than 2a and 2b; the remaining three compounds were not active against P. cynomolgi. All five compounds showed much higher blood schizonticidal activity and less toxicity than primaquine; however, none of the compounds were as active as 2a. Three of four compounds tested showed high activity against P. berghei yoelii.


Subject(s)
Malaria/drug therapy , Primaquine/analogs & derivatives , Animals , Chemical Phenomena , Chemistry , Macaca mulatta , Mice , Plasmodium , Primaquine/chemical synthesis , Primaquine/therapeutic use , Structure-Activity Relationship
16.
J Med Chem ; 23(5): 581-4, 1980 May.
Article in English | MEDLINE | ID: mdl-6770089

ABSTRACT

A series of 2,4-disubstituted 8-aminoquinoline analogues were synthesized and evaluated against Plasmodium berghei in mice and Leishmania donovani in hamsters. 8-[[6-(Diethylamino)hexyl]amino]-2-ethyl-6-methoxy-4-methylquinoline (8a) possessed significant activity against L. donovani. 2-Ethyl-4-methylprimaquine (7a) was evaluated against Plasmodium cynomolgi in rhesus monkey and found to have activity equal to that of primaquine.


Subject(s)
Aminoquinolines/chemical synthesis , Antiprotozoal Agents/chemical synthesis , Aminoquinolines/pharmacology , Animals , Antimalarials/chemical synthesis , Chemical Phenomena , Chemistry , Haplorhini , Leishmania/drug effects , Macaca mulatta , Malaria/drug therapy , Plasmodium/drug effects
17.
J Med Chem ; 22(11): 1363-7, 1979 Nov.
Article in English | MEDLINE | ID: mdl-118257

ABSTRACT

4(beta-Alkylvinyl)-6-methoxy-8-nitroquinolines (6) were prepared from 6-methoxy-8-nitroquinoline-4-carboxaldehyde (5) via a Wittig reaction. Stannous chloride reduction of 6 gave 4-(beta-alkylvinyl)-8-amino-6-methoxyquinolines (8), whereas catalytic reduction of 6 using Raney nickel catalyst gave 4-alkyl-8-amino-6-methoxyquinolines (7). Alkylation of 7 and 8 with 4-iodo-1-phthalimidopentane, followed by removal of the phthaloyl-protecting group with hydrazine, gave 4-alkyl and 4-(beta-alkylvinyl) derivatives of primiquine, respectively. These compounds were evaluated for antimalarial activity against P. berghei and P. berghei yoelii in mice and against P. cynomolgi in rhesus monkeys. Several of the compounds were active in the P. bergheii yoelii screen. None of the compounds showed significant activity in the other two screens.


Subject(s)
Primaquine/analogs & derivatives , Animals , Haplorhini , Macaca mulatta , Malaria/prevention & control , Mice , Plasmodium , Plasmodium berghei , Primaquine/chemical synthesis
18.
J Med Chem ; 22(6): 694-9, 1979 Jun.
Article in English | MEDLINE | ID: mdl-110932

ABSTRACT

The 4-vinyl, 4-ethyl, and three 4-[beta-(arylthio)ethyl] derivatives of primaquine and other 8-aminoquinoline antimalarial agents were prepared for antimalarial evaluation. 8-[(4'-Amino-1'-methylbutyl)amino]-4-ethyl-6-methoxyquinoline (4-ethylprimaquine), which showed activity approximately equal to that of primaquine against Plasmodia cynomolgi in Rhesus monkey, was the most active of the compounds tested. 4-Ethylprimaquine was also less toxic than primaquine, as measured in the Rane mouse screen.


Subject(s)
Aminoquinolines/chemical synthesis , Antimalarials/chemical synthesis , Aminoquinolines/pharmacology , Aminoquinolines/therapeutic use , Animals , Antimalarials/therapeutic use , Dose-Response Relationship, Drug , Haplorhini , Macaca mulatta , Malaria/drug therapy , Malaria/prevention & control , Mice , Plasmodium berghei , Primaquine/therapeutic use
19.
J Med Chem ; 21(4): 326-30, 1978 Apr.
Article in English | MEDLINE | ID: mdl-349156

ABSTRACT

The resolution of several antimalarial agents via pi-complex formation with alpha-(2,4,5,7-tetranitro-9-fluorenylideneaminooxy) propionic acid (TAPA) is reported. Since this represents the first use of this agent for the resolution of amines, some details of the separations are presented. The method proved successful for resolving weakly alkaline amines that did not form stable salts with common resolving acids, highly insoluble amines that did not form soluble salts with usual resolving acids, and amines that did not form crystalline salts with commonly available resolving acids. The optical isomers of several antimalarial agents were evaluated against Plasmodium berghei in the mouse. None of the optically active forms showed any significant differences. The curative activity of (+)- and (-)-primaquine against Plasmodium cynomolgi in the rhesus monkey was essentially identical; however, significant differences in toxicity were noted.


Subject(s)
Antimalarials/isolation & purification , Fluorenes , Animals , Antimalarials/therapeutic use , Malaria/drug therapy , Methods , Mice , Optical Rotation , Plasmodium berghei , Propionates , Stereoisomerism
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