Comparison of in-situ hybridization, direct and indirect in-situ PCR as well as tyramide signal amplification for the detection of HPV

 One hundred paraffin-embedded cervical biopsy specimens were tested for the presence of human papilloma virus (HPV) by in situ hybridization (ISH), and by direct and indirect in situ PCR (IS-PCR) in order to evaluate the efficiency of the different in situ methods in detecting HPV infection. ISH was performed using either commercial DNA probes or a cocktail of 5′-digoxigenin labeled oligoprimers. The same were used for ISH during indirect IS-PCR. To enhance the sensitivity of ISH several polymers, i.e., polyvinyl alcohol (PVA), polyethylene glycol, and polyvinylpyrrolidone were added to the alkaline phosphatase nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) reaction. Furthermore, tyramide signal amplification (TSA) was tried for signal amplification. Those samples treated with PVA during the NBT/BCIP reaction did not show any signal amplification whereas those treated with TSA exhibited a dramatic increase in sensitivity with usually acceptable signal to noise ratios. Our results show that, regarding sensitivity, ISH with subsequent signal amplification by TSA can be used as an almost equivalent alternative to the more cumbersome IS-PCR on routinely processed tissue specimens. When considering reproducibility, it is superior to IS-PCR. Accepted: 25 September 1998     

Visual detection of labeled oligonucleotides using visible-light-polymerization-based amplification

DNA biochip technology holds potential for highly parallel, rapid, and sensitive genetic diagnostic screening of target pathogens and disease biomarkers. A primary limitation involves a simultaneous, sequence-specific identification of low copy number target polynucleotides using a clinically appropriate detection methodology that implements only inexpensive detection instrumentation. Here, a rapid (20 min), nonenzymatic method of signal amplification utilizing surface-initiated photopolymerization is presented in glass microarray format. Visible light photoinitiators covalently coupled to streptavidin were used to bind biotin-labeled capture sequences. Amplification was achieved through subsequent contact with a monomer solution and the appropriate light exposure to generate 20-240-nm-thick hydrogel layers exclusively from spots containing the biotin-labeled DNA. An amplification factor of 10(6) to 10(7) was observed as well as a detectable response generated from as low as approximately 10(4) labeled oligonucleotides using minimal instrumentation, such as an optical microscope or CCD camera.     

A closed tube format for amplification and detection of DNA based on energy transfer.

A new method for the direct detection of PCR-amplified DNA in a closed system is described. The method is based on the incorporation of energy transfer-labeled primers into the amplification product. The PCR primers contain hairpin structures on their 5'ends with donor and acceptor moieties located in close proximity on the hairpin stem. The primers are designed in such a way that a fluorescent signal is generated only when the primers are incorporated into an amplification product. A signal to background ratio of 35:1 was obtained using the hairpin primers labeled with fluorescein as a donor and 4-(4'-dimethylaminophenylazo) benzoic acid (DABCYL) as a quencher. The modified hairpin-primers do not interfere with the activity of DNA polymerase, and both thermostable Pfu and Taq polymerase can be used. This method was applied to the detection of cDNA for prostate specific antigen. The results demonstrate that the fluorescent intensity of the amplified product correlates with the amount of incorporated primers, and as few as 10 molecules of the initial template can be detected. This technology eliminates the risk of carry-over contamination, simplifies the amplification assay and opens up new possibilities for the real-time quantification of the amplified DNA over an extremely wide dynamic range.     

Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol

The telomeric repeat amplification protocol (TRAP) is a two-step process for analyzing telomerase activity in cell or tissue extracts. Recent modifications of this sensitive assay include elimination of radioactivity by using a fluorescently labeled primer instead of a radiolabeled primer. In addition, the TRAP assay has been modified for real-time, quantitative PCR analysis. Here, we describe cost-effective procedures for detection of telomerase activity using a fluorescent-based assay as well as by using real-time PCR. These modified TRAP assays can be accomplished within 4 h (from lysis of samples to analysis of telomerase products).     

Mobility measurements of immunomagnetically labeled cells allow quantitation of secondary antibody binding amplification.

Magnetic cell separation methods commonly utilize paramagnetic materials conjugated to antibodies that target specific cell surface molecules. The amount of magnetic material bound to a cell is directly proportional to the magnetophoretic mobility of that cell. A mathematical model has been developed which characterizes the fundamental parameters controlling the amount of magnetic material bound, and thus, the magnetophoretic mobility of an immunomagnetically labeled cell. In characterization of the paramagnetic labeling, one of the parameters of interest is the increase in magnetophoretic mobility due to the secondary antibody binding to multiple epitopes on the primary antibody, referred to as the "secondary antibody binding amplification," Psi. Secondary antibody-binding amplification has been investigated and quantitated by comparing the mobilities of lymphocytes directly labeled with anti-CD4 MACS (Miltenyi Biotec, Auburn, CA) magnetic nanoparticle antibody with the mobilities of lymphocytes from the same sample labeled with two different indirect antibody-labeling schemes. Each indirect labeling scheme incorporated a primary mouse anti-CD4 FITC antibody that provides both FITC and mouse-specific binding sites for two different secondary antibody-magnetic nanoparticle conjugates: either anti-FITC MACS magnetic nanoparticle antibody or anti-mouse MACS magnetic nanoparticle antibody. The magnetophoretic mobilities of the immunomagnetically labeled cells were obtained using Cell Tracking Velocimetry (CTV). The results indicate that an average of 3.4 anti-FITC MACS magnetic nanoparticle antibodies bind to each primary CD4 FITC antibody, Psi(1,2f) = 3.4 +/- 0.33, and that approximately one, Psi(1,2m) = 0.98 +/- 0.081, anti-mouse MACS magnetic nanoparticle antibody binds to each primary mouse CD4 FITC antibody on a CD4 positive lymphocyte. These results have provided a better understanding of the antibody-binding mechanisms used in paramagnetic cell labeling for magnetic cell separation.     

PCR amplification on a microarray of gel-immobilized oligonucleotides: detection of bacterial toxin- and drug-resistant genes and their mutations

PCR amplification on a microarray of gel-immobilized primers (microchip) has been developed. One of a pair of PCR primers was immobilized inside a separate microchip polyacrylamide porous gel pad of 0.1 x 0.1 x 0.02 (or 0.04) micron in size and 0.2 (or 0.4) nL in volume. The amplification was carried out simultaneously both in solution covering the microchip array and inside gel pads. Each gel pad contained the immobilized forward primers, while the fluorescently labeled reverse primers, as well as all components of the amplification reaction, diffused into the gel pads from the solution. To increase the amplification efficiency, the forward primers were also added into the solution. The kinetics of amplification was measured in real time in parallel for all gel pads with a fluorescent microscope equipped with a charge-coupled device (CCD) camera. The accuracy of the amplification was assessed by using the melting curves obtained for the duplexes formed by the labeled amplification product and the gel-immobilized primers during the amplification process; alternatively, the duplexes were produced by hybridization of the extended immobilized primers with labeled oligonucleotide probes. The on-chip amplification was applied to detect the anthrax toxin genes and the plasmid-borne beta-lactamase gene responsible for bacterial ampicillin resistance. The allele-specific type of PCR amplification was used to identify the Shiga toxin gene and discriminate it from the Shiga-like one. The genomic mutations responsible for rifampicin resistance of the Mycobacterium tuberculosis strains were detected by the same type of PCR amplification of the rpoB gene fragment isolated from sputum of tuberculosis patients. The on-chip PCR amplification has been shown to be a rapid, inexpensive and powerful tool to test genes responsible for bacterial toxin production and drug resistance, as well as to reveal point nucleotide mutations.     

PCR amplification and simultaneous digoxigenin incorporation of long DNA probes for fluorescence in situ hybridization.

Nonradioactive in situ hybridization has found widespread applications in cytogenetics. Basic requirements are DNA probes in sufficient amounts and of high specificity as well as a labeling protocol of good reproducibility. The PCR has been of fundamental importance for the amplification of DNA sequences and thus for the production of DNA probes. Meanwhile, PCR protocols for amplification of DNA have reached a high degree of automation. So far, incorporation of labeled nucleotides into these DNA probes has normally been done by nick translation. Here we show that in using the PCR, amplification of a DNA probe larger than one kilobase accompanied by simultaneous incorporation of digoxigenin-11-dUTP can be performed for in situ hybridization experiments. As an example, the DNA probe pUC 1.77 specific for the subcentromeric region q12 of chromosome number 1 was used and hybridized against metaphase chromosomes from human lymphocytes. The labeled chromosome region was detected by anti-digoxigenin-fluorescein, Fab fragments. The experiments were evaluated by digital image analysis of microphotographs.     

Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms

Different chemical methods used to attach oligonucleotides by their 5′-end on a glass surface were tested in the framework of solid phase PCR where surface-bound instead of freely-diffusing primers are used to amplify DNA. Each method was first evaluated for its capacity to provide a high surface coverage of oligonucleotides essentially attached via a 5′-specific linkage that satisfyingly withstands PCR conditions and leaves the 3′-ends available for DNA polymerase activity. The best results were obtained with 5′-thiol-modified oligonucleotides attached to amino-silanised glass slides using a heterobifunctional cross-linker reagent. It was then demonstrated that the primers bound to the glass surface using the optimal chemistry can be involved in attaching and amplifying DNA molecules present in the reaction mix in the absence of freely-diffusing primers. Two distinct amplification processes called interfacial and surface amplification have been observed and characterised. The newly synthesised DNA can be detected and quantified by radioactive and fluorescent hybridisation assays. These new surface amplification processes are seen as an interesting approach for attachment of DNA molecules by their 5′-end on a solid support and can be used as an alternative route for producing DNA chips for genomic studies.     

Oligo(dA-dT)-dependent signal amplification for the detection of proteins in cells

An ultrasensitive protein detection system in situ named the ImmunoAT-tailing method was developed. It consists of three elementary processes: (i) detection of a protein by a primary antibody and a biotinylated secondary antibody; (ii) linking of biotinylated 15-base oligo(dA-dT) to the biotinylated immunocomplex via streptavidin; and (iii) self-priming elongation of oligo(dA-dT) by the Klenow fragment, 3' to 5' exo-. After the elongation reaction in the presence of dATP, dTTP, and dye-labeled dUTP, the protein was labeled with a large number of the dye molecules. The poly(dA-dT) elongated without the labeled nucleotides was detected by 4',6-diamidino-2-phenylindole (DAPI) staining. By combining the different labelings, double staining was possible. This ImmunoAT-tailing method has a specificity and sensitivity higher than that of tyramide signal amplification.     

cRNA target preparation for microarrays: comparison of gene expression profiles generated with different amplification procedures

Microarray technology has become a standard tool for generation of gene expression profiles to explore human disease processes. Being able to start from minute amounts of RNA extends the fields of application to core needle biopsies, laser capture microdissected cells, and flow-sorted cells. Several RNA amplification methods have been developed, but no extensive comparability and concordance studies of gene expression profiles are available. Different amplification methods may produce differences in gene expression patterns. Therefore, we compared profiles processed by a standard microarray protocol with three different types of RNA amplification: (i) two rounds of linear target amplification, (ii) random amplification, and (iii) amplification based on a template switching mechanism. The latter two methods accomplish target amplification in a nonlinear way using PCR technology. Starting from as little as 50 ng of total RNA, the yield of labeled cRNA was sufficient for hybridization to Affymetrix HG-U133A GeneChip array using the respective methods. Replicate experiments were highly reproducible for each method. In comparison with the standard protocol, all three approaches are less sensitive and introduced a minor but clearly detectable bias of the detection call. In conclusion, the three amplification protocols used are applicable for GeneChip analysis of small tissue samples.     

Reciprocating-flow ATP amplification system for increasing the number of amplification cycles

We constructed a novel ATP amplification reactor using a reciprocating-flow system to increase the number of ATP amplification cycles without an increase in backpressure. We previously reported a continuous-flow ATP amplification system that effectively and quantitatively amplified ATP and increased the sensitivity of a quantitative bioluminescence assay. However, it was difficult to increase the number of amplification cycles due to backpressure in the system. Because addition of immobilized adenylate kinase (ADK) and pyruvate kinase (PK) columns increased backpressure, the maximum number of ATP amplification cycles within column durability was only 4. In this study, ATP amplification was performed using a reciprocating-flow system, and 10 cycles of ATP amplification could be achieved without an increase in backpressure. As a result, ATP was amplified more than 100-fold after 10 cycles of reciprocating flow. The gradient of ATP amplification was approximately 1.76^N. The backpressure on the columns was 0.03 MPa in 1–10 ATP amplification cycles, and no increases in backpressure were observed.     

Sensitive and isothermal electrochemiluminescence gene-sensing of Listeria monocytogenes with hyperbranching rolling circle amplification technology

Listeria monocytogenes (L. monocytogenes) is one of the most problematic human pathogens, as it is mainly transmitted through the food chain and cause listeriosis. Thus, specific and sensitive detection of L. monocytogenes is required to ensure food safety. In this study, we proposed a method using hyperbranching rolling circle amplification (HRCA) combined with magnetic beads based electrochemiluminescence (ECL) to offer an isothermal, highly sensitive and specific assay for the detection of L. monocytogenes. At first, a linear padlock probe was designed to target a specific sequence in the hly gene which is specific to L. monocytogenes and then ligated by Taq DNA ligase. After ligation and digestion, further amplification by HRCA with a biotiny labeled primer and a tris (bipyridine) ruthenium (TBR) labeled primer was performed. The resulting HRCA products were then captured onto streptavidin-coated paramagnetic beads and were analyzed by magnetic beads based ECL platform to confirm the presence of targets. Through this approach, as low as 10 aM synthetic hly gene targets and about 0.0002 ng/μl of genomic DNA from L. monocytogenes can be detected, the ability to detect at such ultratrace levels could be attributed to the powerful amplification of HRCA and the high sensitivity of current magnetic bead based ECL detection platform.     

Two-color,rolling-circle amplification on antibody microarrays for sensitive,multiplexed serum-protein measurements

The ability to conveniently and rapidly profile a diverse set of proteins has valuable applications. In a step toward further enabling such a capability, we developed the use of rolling-circle amplification (RCA) to measure the relative levels of proteins from two serum samples, labeled with biotin and digoxigenin, respectively, that have been captured on antibody microarrays. Two-color RCA produced fluorescence up to 30-fold higher than direct-labeling and indirect-detection methods using antibody microarrays prepared on both polyacrylamide-based hydrogels and nitrocellulose. Replicate RCA measurements of multiple proteins from sets of 24 serum samples were highly reproducible and accurate. In addition, RCA enabled reproducible measurements of distinct expression profiles from lower-abundance proteins that were not measurable using the other detection methods. Two-color RCA on antibody microarrays should allow the convenient acquisition of expression profiles from a great diversity of proteins for a variety of applications.     

Ferrocenemonocarboxylic-HRP@Pt nanoparticles labeled RCA for multiple amplification of electro-immunosensing

A multiple amplification immunoassay was proposed to detect alpha-fetoprotein (AFP), which was based on ferrocenemonocarboxylic-HRP conjugated on Pt nanoparticles as labels for rolling circle amplification (RCA). Firstly, the capture antibody (anti-AFP) was immobilized on glass carbon electrode (GCE) deposited nano-sized gold particles. After a typical immuno-sandwich protocol, primary DNA was immobilized by labeling secondary antibody, which acted as a precursor to initiate RCA. The products of RCA provide large amount of sites to link detection DNAs, which were labeled by signal probes (ferrocenemonocarboxylic) and horseradish peroxidase (HRP). Moreover, the enzymatic amplification signals could be produced by the catalysis of HRP and Pt nanoparticles with the addition of H?O?. These lead to multiple amplification signals monitoring by electrochemical instrument and further resulted in high sensitivity of the immunoassay with the detection limit of 1.7 pg/mL.     

Limited efficiency of universal mini-barcode primers for DNA amplification from desert reptiles, birds and mammals

In recent years, DNA barcoding has emerged as a powerful tool for species identification. We report an extended validation of a universal DNA mini-barcode for amplification of 130-bp COI segments from 23 specimens collected from a desert environment, including 11 reptiles, five mammals and seven birds. Besides the standard double-annealing protocol, we also tested a more stringent single-annealing protocol. The PCR success rate for the amplification of the mini-barcode region was: mammals (4/5), reptiles (5/11) and birds (4/7). These findings demonstrate the limited utility of universal primers for mini-barcoding, at least for these vertebrate taxa that we collected from the Saudi Arabian desert.     

Direct genomic fluorescent on-line sequencing and analysis using in vitro amplification of DNA.

In vitro amplification of genomic DNA and total RNA, as well as recombinant DNA, using one fluorescently labelled and one unlabelled primer during amplification, together with on-line analysis of the products on the EMBL fluorescent DNA sequencer, is described. Further is reported direct sequencing of fluorescently labelled amplified probes by solid-phase chemical degradation, without subcloning and purification steps involved. At present up to 350 bases in 4 hours are determined with this technique. The fluorescent dye and its bond to the oligonucleotide are stable during the amplification cycles, and do not interfere with the enzymatic polymerization. High sensitivity of the detection device, down to 10(-18) moles, corresponding to less than 10(6) molecules makes possible analyses of the non-radioactive amplified probes after only 10 amplification cycles, starting with about 5 x 10(4) copies of recombinant DNA.     

TempliPhi,phi29 DNA polymerase based rolling circle amplification of templates for DNA sequencing

We have developed a novel, isothermal DNA amplification strategy that employs phi29 DNA polymerase and rolling circle amplification to generate high-quality templates for DNA sequencing reactions. The TempliPhi DNA amplification kits take advantage of the fact that cloned DNA is typically obtained in circular vectors, which are readily replicated in vitro using phi29 DNA polymerase by a rolling circle mechanism. This single subunit, proofreading DNA polymerase has excellent processivity and strand displacement properties for generation of multiple, tandem double-stranded copies of the circular DNA, generating as much as 10(7)-fold amplification. Large amounts of product (1-3 microg) can be obtained in as little as 4 hours. Input DNA can be as little as 0.01 ng of purified plasmid DNA, a single bacterial colony, or a 1 microL of a saturated overnight culture. Additionally, the presence of an associated proof reading function within the phi29 DNA polymerase ensures high-fidelity amplification. Once completed, the product DNA can be used directly in sequencing reactions. Additionally, the properties of phi29 DNA polymerase and its use in applications such as amplification ofhuman genomic DNA for genotyping studies is discussed.