A two-step hybridization method for chemiluminescent detection of single copy genes.

We have developed a technique for the chemiluminescent detection of single copy genes that eliminates the high backgrounds and problems with probe labeling associated with existing methods. The procedure employs a primary hybridization of single-stranded probe DNA to immobilized target DNA, a secondary hybridization with a covalently cross-linked oligonucleotide-alkaline phosphatase conjugate, followed by incubation in the chemiluminescent substrate AMPPD and detection on x-ray film. The key to the success of this method is that the primary probe contains a region complementary to the target DNA as well as to the oligonucleotide sequence of the secondary probe-alkaline phosphatase conjugate. Here we report our results using the two-step hybridization procedure to detect single copy genes from genomic Southern blots.     

Theoretical aspects of genomic variation screening using DNA microarrays

We present a theoretical model for typical microarray-based single nucleotide polymorphism (SNP) assay of small genomic DNA amount. We derived the adsorption isotherm expressing the on-array hybridization efficiency in terms of genomic target sequence and concentration, oligonucleotide probe sequence and surface density, hybridization buffer, and temperature. This isotherm correctly describes the surface probe density effects, the sensitivity peak, and the melting temperature depression, and is in accord with published experiments. We discuss optimization of parallel SNP genotyping. Our estimates show that SNP detection at a single temperature in aqueous hybridization buffer is restricted by DNA regions that differ by less than 20% in GC content. We predict that the variety of genotyped SNPs could be substantially extended using an assay design with high probe density and a large fraction of probes hybridized.     

Genomic Southern analysis with alkaline-phosphatase-conjugated oligonucleotide probes and the chemiluminescent substrate AMPPD

We have used a chemiluminescent detection method to improve both the sensitivity and the speed of detection of human genes with oligonucleotide probes. A direct chemiluminescent substrate (AMPPD) was used in combination with an alkaline-phosphatase-labeled oligonucleotide probe to detect the human tissue of plasminogen activator gene by Southern blot analysis. X-ray exposures obtained after 4 h were comparable to those obtained after 7 days with a 32P-labeled oligomer. After 16 h, the signal was 12 times greater than the 32P signal. The detection of the single-copy tissue plasminogen activator gene in 0.25 micrograms of human genomic DNA (76,000 molecules) was achieved. The improved sensitivity obtained by chemiluminescent detection should increase the usefulness of oligonucleotide probes in the direct Southern analysis of human genetic disorders.     

Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate.

This study evaluates a DNA hybridization assay for salmonella with AttoPhos (JBL Scientific, San Luis Obispo, CA), a fluorescent substrate for alkaline phosphatase. The probe used (50 ng/ml) was a biotinylated 600 bp fragment consisting of a tandem repeat of an insertion sequence (IS200) found in most Salmonella spp. evaluated. The hybridization was carried out at 65 degrees C for 2 h without prior prehybridization and hybrids were detected by the addition of a streptavidin-alkaline phosphatase conjugate. Circles (5 mm) were cut from the membrane and placed in a cuvette containing 1 ml of 1 mmol/l AttoPhos. The reaction was evaluated after 30 min at 37 degrees C with a fluorometer with an excitation wavelength of 440 nm and an emission wavelength of 550 nm. The sensitivity of the probe was estimated to be 10,000 copies of target DNA or 5 x 10(-20) mol of DNA. All 74 salmonella strains tested reacted with the probe but none of the 98 heterologous species tested gave positive results. The results of this study indicate that our assay method, which employs a biotinylated tandem repeat of IS200 and AttoPhos, is a specific and highly sensitive quantitative method for the detection of salmonellas.     

A direct repeat sequence associated with the centromeric retrotransposons in wheat.

A high-density BAC filter of Triticum monococcum was screened for the presence of a centromeric retrotransposon using the integrase region as a probe. Southern hybridization to the BAC digests using total genomic DNA probes of Triticum monococcum, Triticum aestivum, and Hordeum vulgare detected differentially hybridizing restriction fragments between wheat and barley. The fragments that hybridized to genomic DNA of wheat but not to that of barley were subcloned. Fluorescence in situ hybridization (FISH) analysis indicated that the clone pHind258 hybridized strongly to centromeric regions in wheat and rye and weakly to those in barley. The sequence of pHind258 was homologous to integrase and long terminal repeats of centromeric Ty3-gypsy retrotransposons of cereal species. Additionally, pHind258 has a pair of 192-bp direct repeats. FISH analysis indicated that the 192-bp repeat probe hybridized to centromeres of wheat and rye but not to those of barley. We found differential FISH signal intensities among wheat chromosomes using the 192-bp probe. In general, the A-genome chromosomes possess strong FISH signals, the B-genome chromosomes possess moderate signals, and the D-genome chromosomes possess weak signals. This was consistent with the estimated copy numbers of the 192-bp repeats in the ancestral species of hexaploid wheat.     

A novel endonuclease IV post-PCR genotyping system

Here we describe a novel endonuclease IV (Endo IV) based assay utilizing a substrate that mimics the abasic lesions that normally occur in double-stranded DNA. The three component substrate is characterized by single-stranded DNA target, an oligonucleotide probe, separated from a helper oligonucleotide by a one base gap. The oligonucleotide probe contains a non-fluorescent quencher at the 5′ end and fluorophore attached to the 3′ end through a special rigid linker. Fluorescence of the oligonucleotide probe is efficiently quenched by the interaction of terminal dye and quencher when not hybridized. Upon hybridization of the oligonucleotide probe and helper probe to their complementary target, the phosphodiester linkage between the rigid linker and the 3′ end of the probe is efficiently cleaved, generating a fluorescent signal. In this study, the use of the Endo IV assay as a post-PCR amplification detection system is demonstrated. High sensitivity and specificity are illustrated using single nucleotide polymorphism detection.     

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY((R)) FL-labeled probe or primer

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY((R)) FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY((R)) FL-modified cytosine at its 5'-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.     

Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis

Abstract A 277-bp long DNA fragment, Ba813, was isolated from an avirulent Bacillus anthracis strain 7700 genomic library. Two oligonucleotides derived from the Ba813 sequence were used as primers in polymerase chain reaction tests on genomic DNA from 28 Bacillus anthracis and from 33 heterologous bacteria strains. A specific, 152-bp long DNA fragment was amplified only when Bacillus anthracis DNA was used as the target. The amplified product was analysed by non-radioactive sandwich hybridisation in microtiter plates using two oligonucleotides. The capture oligonucleotide C1 was covalently linked onto aminated wells of microtiter plates. The detection oligonucleotide D3 was labelled with biotine. The hybrid molecules were detected by avidine conjugated with alkaline phosphatase and chromogenic substrate. Amplification of Ba813 sequence may provide the basis for rapid and reliable assay for the detection and identification of Bacillus anthracis .     

Use of an arbitrarily primed PCR product in the development of a Campylobacter jejuni-specific PCR.

Development of a PCR assay for Campylobacter jejuni is based on the isolation of species-specific DNA. An arbitrarily primed PCR incorporating 10-mer primers was used to generate fingerprints of C. jejuni M129 genomic DNA. Fingerprint products were then screened individually for their species specificity in dot blot hybridizations with 6 C. jejuni isolates, 4 Campylobacter species other than C. jejuni, and 27 enteric bacterial species other than Campylobacter spp. A 486-bp fingerprint product hybridized specifically to C. jejuni DNA under stringent conditions; no binding to Campylobacter DNA other than that of C. jejuni or to DNA from enteric bacteria was detected. The 486-bp fingerprint product was sequenced, and primers corresponding to three overlapping regions of the DNA probe were synthesized. Evaluation of the three primer pairs for specificity to C. jejuni DNA identified an oligonucleotide primer pair which amplified a 265-bp product from six C. jejuni isolates only. In sensitivity studies using a crude M129 lysate as the template, the C. jejuni-specific PCR amplified the 265-bp product in a lysate with as few as 100 bacteria.     

Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid

The hybridization behavior of small oligonucleotides arrayed on glass slides is currently unpredictable. In order to examine the hybridization efficiency of capture probes along target nucleic acid, 20-mer oligonucleotide probes were designed to hybridize at different distances from the 5' end of two overlapping 402- and 432-bp ermB products amplified from the target DNA. These probes were immobilized via their 5' end onto glass slides and hybridized with the two labeled products. Evaluation of the hybridization signal for each probe revealed an inverse correlation with the length of the 5' overhanging end of the captured strand and the hybridization signal intensity. Further experiments demonstrated that this phenomenon is dependent on the reassociation kinetics of the free overhanging tail of the captured DNA strand with its complementary strand. This study delineates key predictable parameters that govern the hybridization efficiency of short capture probes arrayed on glass slides. This should be most useful for designing arrays for detection of PCR products and nucleotide polymorphisms.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe.

A single synthetic oligonucleotide was employed as hybridization probe to detect and enable isolation of the human insulin-like growth factor I (IGF-I) gene from a human genomic DNA library. The synthetic oligonucleotide probe coded for the B-chain of IGF-I and was designed for expression in Escherichia coli. Despite numerous interspersed mismatches, the synthetic probe hybridized specifically with seven recombinant lambda phage containing almost the entire B-chain region of the human IGF-I gene. The usefulness of this approach was further demonstrated by the detection of lambda phage containing human preproinsulin, using A and B chain synthetic oligonucleotides, 90 and 63 nucleotides in length, as hybridization probes. The nucleotide sequence of the human IGF-I exon suggests that IGF-I is synthesized as a larger precursor molecule.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Use of an oligonucleotide probe to detect Vibrio parahaemolyticus in artificially contaminated oysters.

A 26-mer oligonucleotide specific to Vibrio parahaemolyticus was synthesized from a 1,275-bp thermostable direct hemolysin (tdh) gene. This oligonucleotide probe specifically reacted with DNA from 89 of 95 V. parahaemolyticus isolates but not with DNA from other vibrios or other enteric and nonenteric organisms (n = 48). The probe hybridized with Southern blots of 0.5-kb HindIII-restricted chromosomal DNA fragments from all but five V. parahaemolyticus test isolates. The probe could be used to directly identify V. parahaemolyticus in artificially contaminated food without an isolation step.     

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.     

Fluorescent quenching-based quantitative detection of specific DNA/RNA using a BODIPY® FL-labeled probe or primer

We have developed a simple method for the quantitative detection of specific DNA or RNA molecules based on the finding that BODIPY^® FL fluorescence was quenched by its interaction with a uniquely positioned guanine. This approach makes use of an oligonucleotide probe or primer containing a BODIPY^® FL-modified cytosine at its 5′-end. When such a probe was hybridized with a target DNA, its fluorescence was quenched by the guanine in the target, complementary to the modified cytosine, and the quench rate was proportional to the amount of target DNA. This widely applicable technique will be used directly with larger samples or in conjunction with the polymerase chain reaction to quantify small DNA samples.     

A comparison of chemiluminescent and colorimetric substrates in a hepatitis B virus DNA hybridization assay

We compared the sensitivity of a chemiluminescent substrate 3-(2'-spiroadamantane)-4-methoxy-4-(3"-phosphoryloxy)phenyl- 1,2-dioxetane (AMPPD) and a chromogenic substrate 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NBT) for detection of an alkaline phosphatase label in a hepatitis B virus "core antigen" DNA (HBVc) probe hybridization assay. Chemiluminescent signal obtained from AMPPD hydrolysis by alkaline phosphatase was detected with Polaroid Instant Black and White Type 612 film. The chemiluminescent assay detected 1.18 x 10(6) copies of HBVc plasmid DNA in 30 min. By comparison, 9.8 x 10(7) copies of DNA could be measured using chromogenic BCIP/NBT substrate within the same incubation time. After further development, the chemiluminescent endpoint permitted detection of 4.39 x 10(4) copies of HBVc plasmid DNA in 2 h.     

Specific detection of Mycobacterium sp. genomic DNA using dual labeled gold nanoparticle based electrochemical biosensor

The present study was aimed at the development and evaluation of a DNA electrochemical biosensor for Mycobacterium sp. genomic DNA detection in a clinical specimen using a signal amplifier as dual-labeled AuNPs. The DNA electrochemical biosensors were fabricated using a sandwich detection strategy involving two kinds of DNA probes specific to Mycobacterium sp. genomic DNA. The probes of enzyme ALP and the detector probe both conjugated on the AuNPs and subsequently hybridized with target DNA immobilized in a SAM/ITO electrode followed by characterization with CV, EIS, and DPV analysis using the electroactive species para-nitrophenol generated by ALP through hydrolysis of para-nitrophenol phosphate. The effect of enhanced sensitivity was obtained due to the AuNPs carrying numerous ALPs per hybridization and a detection limit of 1.25 ng/ml genomic DNA was determined under optimized conditions. The dual-labeled AuNP-facilitated electrochemical sensor was also evaluated by clinical sputum samples, showing a higher sensitivity and specificity and the outcome was in agreement with the PCR analysis. In conclusion, the developed electrochemical sensor demonstrated unique sensitivity and specificity for both genomic DNA and sputum samples and can be employed as a regular diagnostics tool for Mycobacterium sp. monitoring in clinical samples.     

Dramatic increase in the signal and sensitivity of detection <Emphasis Type="Italic">via</Emphasis> self-assembly of branched DNA

In molecular testing using PCR, the target DNA is amplified via PCR and the sequence of interest is investigated via hybridization with short oligonucleotide capture probes that are either in a solution or immobilized on solid supports such as beads or glass slides. In this report, we report the discovery of assembly of DNA complex(es) between a capture probe and multiple strands of the PCR product. The DNA complex most likely has branched structure. The assembly of branched DNA was facilitated by the product of asymmetric PCR. The amount of branched DNA assembled was increased five fold when the asymmetric PCR product was denatured and hybridized with a capture probe all in the same PCR reaction mixture. The major branched DNA species appeared to contain three reverse strands (the strand complementary to the capture probe) and two forward strands. The DNA was sensitive to S1 nuclease suggesting that it had single-stranded gaps. Branched DNA also appeared to be assembled with the capture probes immobilized on the surface of solid support when the product of asymmetric PCR was hybridized. Assembly of the branched DNA was also increased when hybridization was performed in complete PCR reaction mixture suggesting the requirement of DNA synthesis. Integration of asymmetric PCR, heat denaturation and hybridization in the same PCR reaction mixture with the capture probes immobilized on the surface of solid support achieved dramatic increase in the signal and sensitivity of detection of DNA. Such a system should be advantageously applied for development of automated process for detection of DNA.