Determination of prostate specific antigen mRNA in peripheral blood by reverse transcriptase polymerase chain reaction and a simple chemiluminometric hybridization assay in a high-throughput format

Emmanouilidou, Evaggelia; Ioannou, Penelope C.; Christopoulos, Theodore K.; Polizois, Konstantinos

doi:10.1016/s0003-2697(02)00566-3

Cited by 8 publications

(4 citation statements)

References 19 publications

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“…Amplification of Prostate Specific Antigen cDNA. The target DNA was a 233 bp fragment synthesized by amplifying the prostate-specific antigen (PSA) cDNA using the plasmid pcDNA3.PSA as a template. PCR was performed in a total reaction volume of 50 μL consisting of 10 mmol/L Tris-HCl (pH 9.0), 50 mmol/L KCl, 1 g/L Triton X-100, 1.5 mmol/L MgCl 2 , 0.2 mmol/L dNTPs, 25 pmol each of the biotinylated upstream primer (U PSA ) and the downstream primer (D PSA ), 1.25 units of Taq DNA polymerase, and the plasmid.…”

Section: Methodsmentioning

confidence: 99%

Oligonucleotide-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for DNA Analysis by Hybridization

et al. 2003

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The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.

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Section: Methodsmentioning

confidence: 99%

Oligonucleotide-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for DNA Analysis by Hybridization

et al. 2003

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“…The 5 0 -amino-modified (dT) 30 oligonucleotide was conjugated to BSA by using the homobifunctional cross-linking reagent BS 3 as described recently [Emmanouilidou et al, 2003]. Coating of polystyrene microtiter wells was performed with 50 mL of a 5 mg/L solution of BSA-oligonucleotide conjugate in phosphate-buffered saline (PBS) containing 5 mM EDTA for 2 hr at 421C.…”

Section: Conjugation Of (Dt) 30 Oligonucleotide To Albuminmentioning

confidence: 99%

Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension–application to the variants of mannose-binding lectin gene

et al. 2006

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Mannose-binding lectin (MBL) is a key component of the innate immune system, and its deficiency is associated with increased susceptibility to various infections and autoimmune disorders. Since several nucleotide variations in the mannose-binding lectin 2 gene (MBL2) have been associated with the functional deficiency of MBL, there is a growing need to screen its allelic variants and develop genotyping methods for MBL2. In this context we propose a rapid, robust, cost-efficient, and automatable method for detecting all known allelic variants of MBL2. This report introduces for the first time the photoprotein aequorin as a reporter in genotyping by primer extension (PEXT) reactions. The method involves a single PCR amplification of a genomic region that spans all six variant nucleotide sites, i.e., three structural mutations in exon 1 (c.154C>T, pArg52Cys; c.161A>G, p.Gly54Asp; and c.170A>G, p.Gly57Glu), two single nucleotide polymorphisms (SNPs) at positions c.-619G>C and c.-290G>C (promoter region), and one SNP at position c.-66C>T of the 5' untranslated region. PCR is followed by PEXT reactions for each site. Biotin-dUTP is incorporated in the extended primer. The genotyping primers contain a poly(dA) segment at their 5' end. The products are captured by hybridization on the surface of microtiter wells that are coated with a poly(dT)-albumin. The extended primers only are detected by reaction with a streptavidin-aequorin conjugate. The bound photoprotein aequorin is measured within 3 sec by simply adding Ca2+. We carried out extensive optimization studies of the PEXT reaction and genotyped the six nucleotide variant sites using blood specimens from 27 normal DNA samples. The results of the proposed method agreed entirely with the sequencing data.

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“…The authors reported a simple, rapid and sensitive assay protocol for the quantification of PSA mRNA in peripheral blood (a potential marker for molecular staging of prostate cancer) by using an IS, RT-PCR and a chemiluminometric hybridization assay. 40 The key to quantification was the recombinant RNA IS, which had the same primer binding sites and size as the amplified PSA mRNA but differed only in a 24-bp segment. Amplified sequences were labeled with biotin during PCR by using a 5 0 biotinylated upstream primer.…”

Section: Quantitative Pcrmentioning

confidence: 99%

Gene Assays Based on Bio(Chemi)luminescence

Laios

Ioannou²,

Christopoulos

2010

Chemiluminescence and Bioluminescence

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One of the first reports describing the use of recombinant aequorin as a reporter molecule in a bioluminometric nucleic acid hybridization assay was in 1996. 1 Microtiter wells were coated with anti-digoxigenin antibody. The target DNA was hybridized simultaneously with an immobilized digoxigenin-labeled capture probe and a biotinylated detection probe. The hybrids were determined using an aequorin-streptavidin conjugate followed by the measurement of luminescence in the presence of excess Ca 21 (Figure 9.1). The linearity of the assay was in the range 0.1-200 pM (5 amol well À1 to 10 fmol well À1) and the S/B ratio at 0.1 pM (5 amol well À1) was 5.3. A configuration in which the aequorinstreptavidin conjugate was replaced by a preformed complex of biotinylated aequorin to streptavidin resulted in equivalent signals and detectability. An advantage of using the preformed complex is that it was conveniently prepared by simply mixing the two components (streptavidin and biotinylated aequorin), thus providing a practical alternative to the use of aequorin-streptavidin conjugates prepared by covalent crosslinking techniques. The aequorin-biotin and the aequorin-streptavidin conjugates in this study were obtained commercially (the same group later reported the construction of a plasmid suitable for bacterial expression of in vivo-biotinylated aequorin, facilitating further the development of highly sensitive hybridization assays, 2 as well as a method for

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Determination of prostate specific antigen mRNA in peripheral blood by reverse transcriptase polymerase chain reaction and a simple chemiluminometric hybridization assay in a high-throughput format

Cited by 8 publications

References 19 publications

Oligonucleotide-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for DNA Analysis by Hybridization

Oligonucleotide-Functionalized Gold Nanoparticles as Probes in a Dry-Reagent Strip Biosensor for DNA Analysis by Hybridization

Photoprotein aequorin as a novel reporter for SNP genotyping by primer extension–application to the variants of mannose-binding lectin gene

Gene Assays Based on Bio(Chemi)luminescence

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