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Biomedical subjects

G T Walker

Publications and source records attributed to G T Walker.

At least 19 recordsLinked to original sources
PubMed2000

Pathogen analysis and genetic predisposition testing using microelectronic arrays and isothermal amplification.

BACKGROUND: A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies. METHODS: The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background. RESULTS: Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis. CONCLUSIONS: We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.

Bacteria↗
PubMed1998

Quantitative reverse transcription strand displacement amplification: quantitation of nucleic acids using an isothermal amplification technique.

Recent advances in nucleic acid amplification techniques have allowed for quantitation of viral nucleic acid levels in clinical specimens. The most prevalent testing is carried out for HIV viral load. Strand displacement amplification (SDA) is an isothermal DNA amplification system utilizing a restriction enzyme and a DNA polymerase with strand displacement properties. SDA was adapted for quantitative RNA amplification (QRT-SDA) of an HIV gag sequence by including AMV reverse transcriptase, a quantitative control sequence, and 32P-labeled detector oligonucleotides for the HIV and the control sequences. We have also improved the amplification efficiency by including the single-strand binding protein from gene 32 of T4 bacteriophage (T4gp32) to enhance strand displacement replication. In a preliminary analytical demonstration of the technique, RT-SDA was quantitative to within twofold over a range of 500-500,000 transcripts that were generated from a plasmid bearing an HIV gag sequence. QRT-SDA potentially represents a convenient alternative for viral load testing in a clinical setting.

Base Sequence↗
PubMed1997

Simultaneous strand displacement amplification and fluorescence polarization detection of Chlamydia trachomatis DNA.

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.

Base Sequence↗
PubMed1997

Temperature and quenching studies of fluorescence polarization detection of DNA hybridization.

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.

Anisotropy↗
PubMed1996

DNA detection by strand displacement amplification and fluorescence polarization with signal enhancement using a DNA binding protein.

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.

Base Sequence↗
PubMed1996

Detection of M. tuberculosis DNA using thermophilic strand displacement amplification.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying DNA that is based upon the combined action of a DNA polymerase and restriction enzyme. Previously, a form of SDA was developed which utilizes the exonuclease deficient Klenow fragment of E. coli polymerase I (exo Klenow) and the restriction enzyme HincII to achieve 10(8)-fold amplification in 2 h at 37 degrees C (Walker, G.T., 1993, PCR Methods and Applications 3; 1-6). A new thermophilic form of SDA is reported here which uses a restriction endonuclease from Bacillus stearothermophilus (BsoBI) and a 5'-->3' exonuclease deficient polymerase from Bacillus caldotenax (exo Bca). SDA was used to amplify DNA from Mycobacterium tuberculosis. An amplification factor of 10(10)-fold was achieved after 15 min of SDA at 60 degrees C. The new thermophilic system is much more specific than the previous mesophilic system as evidenced by a dramatic decrease in background amplification products. Thermophilic SDA was also optimized with dUTP substituted for TTP to enable amplicon decontamination using uracil-DNA glycosylase.

Bacillus↗
PubMed1996

Strand displacement amplification (SDA) and transient-state fluorescence polarization detection of Mycobacterium tuberculosis DNA.

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.

Base Sequence↗
PubMed1996

Detection of Mycobacterium tuberculosis DNA with thermophilic strand displacement amplification and fluorescence polarization.

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.

DNA Primers↗
PubMed1995

Hybridization of fluorescein-labeled DNA oligomers detected by fluorescence anisotropy with protein binding enhancement.

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.

Base Sequence↗
PubMed1995

A DNA probe assay using strand displacement amplification (SDA) and filtration to separate reacted and unreacted detector probes.

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.

Base Sequence↗
PubMed1994

Multiplex strand displacement amplification (SDA) and detection of DNA sequences from Mycobacterium tuberculosis and other mycobacteria.

Strand Displacement Amplification (SDA) is an isothermal, in vitro method of amplifying a DNA target sequence prior to detection [Walker et al (1992) Nucleic Acids Res., 20, 1691-1693]. Here we describe a multiplex form of SDA that allows two target sequences and an internal amplification control to be co-amplified by a single pair of primers after common priming sequences are spontaneously appended to the ends of target fragments. Multiplex SDA operates at a single temperature, under the same simple protocol previously developed for single-target SDA. We applied multiplex SDA to co-amplification of a target sequence (IS6110) that is specific to members of the Mycobacterium tuberculosis-complex and a target (16S ribosomal gene) that is common to most clinically relevant species of mycobacteria. Both targets are amplified 10(8)-fold during a 2 hour, single temperature incubation. The relative sensitivity of the system was evaluated across a number of clinically relevant mycobacteria and checked for crossreactivity against organisms that are closely related to mycobacteria.

Base Sequence↗
PubMed1993

Chemiluminescent detection of strand displacement amplified DNA from species comprising the Mycobacterium tuberculosis complex.

Strand displacement amplification, a new isothermal in vitro DNA amplification technique, was used to amplify target DNA contained within the IS6110 insertion element of the species within the Mycobacterium complex (Mycobacterium tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti). The target nucleic acid sequence is present in approximately ten, two, one, five and five copies in M. tuberculosis, M. bovis, M. bovis-BCG, M. africanum and M. microti, respectively. Amplified products were detected using a non-isotopic microtitre plate assay employing a biotinylated oligodeoxynucleotide probe and an alkaline phosphatase conjugated oligodeoxynucleotide probe. Lumiphos 530 was the chemiluminescent substrate for alkaline phosphatase. The combination of the strand displacement amplification method with this sensitive and rapid (less than 2 h) detection system resulted in the specific detection of as few as 1-25 initial IS6110 targets in the five Mycobacterium complex species based on signal/noise criteria. Negative results were obtained with eight other Mycobacterium species as well as with 32 non-Mycobacterium species.

Base Sequence↗
PubMed1993

Empirical aspects of strand displacement amplification.

The most attractive feature of SDA is its operation at a single temperature, which removes the need for instrumented temperature cycling as with PCR and the ligase chain reaction. Highly reproducible temperature profiles, over a large array of samples, can burden the accuracy and expense of an amplification technique. However, the expense of a temperature cycler is offset somewhat by the cost of additional enzymes used in isothermal techniques. In comparisons with isothermal, transcription-based techniques, SDA requires fewer enzymes and has a simpler mechanism. SDA may also be more robust than transcription-based processes because it is not susceptible to contaminating ribonuclease activity. This is generally more of a concern when using clinical samples. The most significant disadvantage of SDA is its inability to efficiently amplify long target sequences. Until this short-coming is eliminated, SDA will be assigned to the diagnostic laboratory along with the ligase chain reaction. Currently, SDA cannot compete with PCR in research applications such as the isolation of gene sequences. The second disadvantage of SDA is that it operates at relatively low (nonstringent) temperatures, which produces considerable background reactions. Consequently, SDA reaction products cannot be analyzed routinely by ethidium-stained gel electrophoresis, as is used commonly with PCR, unless the target sample contains a large number of initial targets.

Base Composition↗
PubMed1992

Strand displacement amplification--an isothermal, in vitro DNA amplification technique.

Strand Displacement Amplification (SDA) is an isothermal, in vitro nucleic acid amplification technique based upon the ability of HincII to nick the unmodified strand of a hemiphosphorothioate form of its recognition site, and the ability of exonuclease deficient klenow (exo- klenow) to extend the 3'-end at the nick and displace the downstream DNA strand. Exponential amplification results from coupling sense and antisense reactions in which strands displaced from a sense reaction serve as target for an antisense reaction and vice versa. In the original design (G. T. Walker, M. C. Little, J. G. Nadeau and D. D. Shank (1992) Proc. Natl. Acad. Sci 89, 392-396), the target DNA sample is first cleaved with a restriction enzyme(s) in order to generate a double-stranded target fragment with defined 5'- and 3'-ends that can then undergo SDA. Although effective, target generation by restriction enzyme cleavage presents a number of practical limitations. We report a new target generation scheme that eliminates the requirement for restriction enzyme cleavage of the target sample prior to amplification. The method exploits the strand displacement activity of exo- klenow to generate target DNA copies with defined 5'- and 3'-ends. The new target generation process occurs at a single temperature (after initial heat denaturation of the double-stranded DNA). The target copies generated by this process are then amplified directly by SDA. The new protocol improves overall amplification efficiency. Amplification efficiency is also enhanced by improved reaction conditions that reduce nonspecific binding of SDA primers. Greater than 10(7)-fold amplification of a genomic sequence from Mycobacterium tuberculosis is achieved in 2 hours at 37 degrees C even in the presence of as much as 10 micrograms of human DNA per 50 microL reaction. The new target generation scheme can also be applied to techniques separate from SDA as a means of conveniently producing double-stranded fragments with 5'- and 3'-sequences modified as desired.

Base Sequence↗
PubMed1992

Isothermal in vitro amplification of DNA by a restriction enzyme/DNA polymerase system.

An isothermal in vitro DNA amplification method was developed based upon the following sequence of reaction events. Restriction enzyme cleavage and subsequent heat denaturation of a DNA sample generates two single-stranded target DNA fragments (T1 and T2). Present in excess are two DNA amplification primers (P1 and P2). The 3' end of P1 binds to the 3' end of T1, forming a duplex with 5' overhangs. Likewise, P2 binds to T2. The 5' overhangs of P1 and P2 contain a recognition sequence (5'-GTTGAC-3') for the restriction enzyme HincII. An exonuclease-deficient form of the large fragment of Escherichia coli DNA polymerase I (exo- Klenow polymerase) [Derbyshire, V., Freemont, P. S., Sanderson, M. R., Beese, L., Friedman, J. M., Joyce, C. M. & Steitz, T. A. (1988) Science 240, 199-201] extends the 3' ends of the duplexes using dGTP, dCTP, TTP, and deoxyadenosine 5'-[alpha-thio]triphosphate, which produces hemiphosphorothioate recognition sites on P1.T1 and P2.T2. HincII nicks the unprotected primer strands of the hemiphosphorothioate recognition sites, leaving intact the modified complementary strands. The exo- Klenow polymerase extends the 3' end at the nick on P1.T1 and displaces the downstream strand that is functionally equivalent to T2. Likewise, extension at the nick on P2.T2 results in displacement of a downstream strand functionally equivalent to T1. Nicking and polymerization/displacement steps cycle continuously on P1.T1 and P2.T2 because extension at a nick regenerates a nickable HincII recognition site. Target amplification is exponential because strands displaced from P1.T1 serve as targets for P2 and strands displaced from P2.T2 serve as targets for P1. A 10(6)-fold amplification of a genomic sequence from Mycobacterium tuberculosis or Mycobacterium bovis was achieved in 4 h at 37 degrees C.

Base Sequence↗
PubMed1992

Success and patient satisfaction following the Stamey procedure for stress urinary incontinence.

A long-term followup survey to determine patient satisfaction following Stamey endoscopic vesicourethral suspension was obtained by means of a mail-in questionnaire. A total of 284 charts was reviewed and surveys were mailed, 17 of which were returned for lack of a forwarding address. A total of 192 replies was received for a 72% response rate. Overall improvement was found to be 82%, with approximately half of these patients found to be totally dry. When asked if they would be willing to go through the experience again 65% said they would. Criteria for the success or failure of the procedure were determined, weighing the degree of dryness against the previously mentioned question. With these criteria there was an overall success rate of 73%. Pulmonary disease, body weight greater than 86 kg., prior Marshall-Marchetti-Krantz procedure and concomitant abdominal hysterectomy lowered the success rate. Prior hysterectomy and concomitant vaginal hysterectomy had no influence on the success rate.

Female↗
PubMed1989

Deoxynucleotide-containing oligoribonucleotide duplexes: stability and susceptibility to RNase V1 and RNase H.

Oligoribonucleotide duplexes containing one to four 2'-deoxynucleotide residues were used as substrates for ribonuclease V1 and RNase H. Either deoxyadenosine and/or deoxythymidine were incorporated into the duplex, 5'GGCCGGAUCCGCGC3'-5'GCGCGGAUCCGGCC3' by substitution of the appropriate deoxynucleoside triphosphate into a transcription reaction with T7 RNA polymerase. The melting temperature, Tm, of the duplex (1.8 microM in strands in 50 mM NaCl) containing only ribonucleotides was 79.9 degrees C. Substitution of deoxyadenosine in both strands of the duplex lowered the Tm by 2.4 degrees C. Substitution of deoxythymidine had no measurable effect on the Tm. Comparison of RNase V1 digestion patterns of fully ribonucleotide and deoxy-substituted duplexes suggest that any distortion is localized to the site of the substitution. An oligoribonucleotide containing two deoxy residues directs specific cleavage of RNA by E. coli RNase H. Structural requirements for cleavage are proposed for RNase V1 and RNase H.

Base Sequence↗