Activity Of Taq DNA Polymerase Research Articles

Thyroglobulin may affect telomerase activity in thyroid follicular cells

Telomerase (TA) activity is known to be present in malignant tumor cells, but not in most somatic differentiated cells. TA shows relatively high activity in thyroid cancer cells, but reports vary. This fact prompted us to elucidate whether cell component inhibitors of TA in the thyroid follicles can modulate its activity. The activity of TA extracted from Hela cells was inhibited by mixing with the supernatant fraction of human thyroid tissue extract. To examine the effect of iodine, thyroid hormones (ℓ-T3 and ℓ-T4) and human thyroglobulin (hTg) contained in the thyroid follicles, ℓ-T3, ℓ-T4 and hTg were added to the TRAP assay system in vitro, using TA from Hela cells. Iodine, ℓ-T3 and ℓ-T4 did not affect TA activity, but hTg inhibited the TA activity in a dose-dependent manner (IC50 of hTg: ca 0.45 μM: inhibiting concentration of hTg was from 0.15 μM to 3.0 μM). The hTg inhibition was not evident in the RT-PCR system, suggesting no effect of hTg on Taq DNA polymerase activity. The hTg inhibition of TA activity was attenuated by dNTP but not significantly by TS primer. These data suggest that hTg contained in thyroid follicular cells of various thyroid diseases may affect the TA activity measured in biopsied thyroid specimens, and that the reduction of the TA activity by hTg may induce slow progression and growth, and low grade malignancy of thyroid cancer, particularly differentiated carcinoma.

Rapid Screening Assay for KRAS Mutations by the Modified Smart Amplification Process

Previously, the smart amplification process version 2 (SMAP-2) was developed to detect mutations from tissue and in crude cell lysates and has been used for rapid diagnosis of specific somatic mutations with single-nucleotide precision. The purpose of this study was to develop a rapid and practical method to detect cancer and metastasis in specimens using the SMAP-2 assay. We developed modified SMAP-2 assays that enabled detection of any change in a single codon using a single assay. Rapid SMAP-2 screening assays are suitable for routine clinical identification of critical amino acid substitutions such as codon 12 mutations in KRAS. Primers bracketing the first two nucleotides of KRAS codon 12 were designed so that all possible alleles would be amplified by the SMAP-2 assay. In combination with the peptide nucleic acid (PNA) with exact homology to the wild-type allele, our assay amplified all mutant alleles except for the wild-type sequence. With this new assay design (termed PNA-clamp SMAP-2), we could detect KRAS mutations within 60 minutes, including sample preparation. We compared results from PNA-clamp SMAP-2 assay, polymerase chain reaction-restriction fragment length polymorphism, and direct sequencing of clinical samples from pancreatic cancer patients and demonstrated perfect concordance. The PNA-clamp SMAP-2 method is a rapid, simple, and highly sensitive detection assay for cancer mutations.

Monitoramento de doença residual mínima em leucemia mielóide crônica por PCR em tempo real

O monitoramento de doença residual mínima (DRM) em leucemia mielóide crônica é extremamente importante, pois possibilita o diagnóstico precoce de eventuais recidivas da doença. Este estudo visa apresentar uma revisão bibliográfica sobre a aplicação e a avaliação da eficácia do método de PCR em tempo real no monitoramento da doença residual mínima em pacientes com leucemia mielóide crônica. O método fornece informações a respeito do número e cinética das células tumorais residuais, sendo atualmente o padrão ouro para seu monitoramento. O uso de transcrição reversa associada à PCR em tempo real tornou a quantificação de mRNA mais simples e precisa. A metodologia mais utilizada é a TaqMan, que emprega a atividade exonucleásica 5'-3' da Taq DNA Polimerase. O monitoramento da DRM é feito após transplante de medula óssea ou após terapias baseadas em drogas, como o interferon-a e o mesilato de imatinibe. Entretanto, os estudos sobre o assunto apresentam os dados de maneiras conflitantes, dificultando a interpretação e comparação dos resultados. A determinação e uniformização dos cut-offs em diversas condições se fazem necessárias a fim de que a metodologia de PCR em tempo real seja aplicada na clínica.

Inhibition of a thermophilic deoxyribonucleic acid polymerase by fullerene derivatives

Enzyme inhibition by fullerene derivatives has attracted much attention. In this communication, effects of two water-solube fullerene derivatives, fullerol and trimalonic acid C60 (TMA C60) on polymerase chain reaction (PCR) were investigated by using PCR of β-actin cDNA derived from HeLa cells as an experimental model. Both fullerol and TMA C60 were found to inhibit PCR in a dose-dependent manner. PCR was ultimately inhibited while the concentrations of each compound were not less than 0.01 mM. In contrast, mannitol exerted no effects on PCR while its concentration increased up to 2 mM. Compensation experiments with Thermus aquaticus (Taq) DNA polymerase revealed that both fullerol and TMA C60 inhibited the enzymatic activity of Taq DNA polymerase, and the inhibitory potency of TMA C60 was slightly greater than that of fullerol. Our data provides some novel aspects on the enzyme inhibiting activities of fullerene derivatives.

Bone mass in young adults: relationship with gender, weight and genetic factors

To determine the relationship of the bone mass attained in young adults with anthropometric and genetic factors. Cross-sectional study of normal individuals. We studied 341 healthy subjects between 22 and 45 years of age. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA) and correlated with body weight, height and nine polymorphisms in six genes involved in sex steroid metabolism (17-hydroxylase, aromatase and 5-reductase) and activity (oestrogen receptors (ER)-alpha and -beta, and androgen receptor). The BMD was higher in men than in women (spine: 1.048 +/- 0.120 vs. 1.034 +/- 0.112; hip: 0.907 +/- 0.131 vs. 0.822 +/- 0.104 g cm(-2), P < 0.001). However, the difference was due, at least in part, to the larger body size in men and diminished markedly after height adjustment. There was a negative correlation between age and hip BMD. Body weight was the single most influential factor on spine and hip BMD in both sexes, explaining 8-9% of BMD variance. Amongst the genetic factors studied, a common CA repeat polymorphism in ER-beta showed a significant association with BMD in women (P = 0.03 at the spine, and 0.008 at the hip). The relationship between ER-beta genotype and BMD persisted after adjustment by body weight and age, explaining a further 2-3% of BMD variance. Allelic variants of other genes studied were not related with BMD. Body weight and allelic variants of ER-beta are associated with BMD in young adults.

Real-Time Polymerase Chain Reaction-Based Approach for Quantification of the pat Gene in the T25 Zea mays Event

In Europe, a growing interest for reliable techniques for the quantification of genetically modified component(s) of food matrixes is arising from the need to comply with the European legislative framework on novel food products. Real-time polymerase chain reaction (PCR) is currently the most powerful technique for the quantification of specific nucleic acid sequences. Several real-time PCR methodologies based on different molecular principles have been developed for this purpose. The most frequently used approach in the field of genetically modified organism (GMO) quantification in food or feed samples is based on the 5'-3'-exonuclease activity of Taq DNA polymerase on specific degradation probes (TaqMan principle). A novel approach was developed for the establishment of a TaqMan quantification system assessing GMO contents around the 1% threshold stipulated under European Union (EU) legislation for the labeling of food products. The Zea mays T25 elite event was chosen as a model for the development of the novel GMO quantification approach. The most innovative aspect of the system is represented by the use of sequences cloned in plasmids as reference standards. In the field of GMO quantification, plasmids are an easy to use, cheap, and reliable alternative to Certified Reference Materials (CRMs), which are only available for a few of the GMOs authorized in Europe, have a relatively high production cost, and require further processing to be suitable for analysis. Strengths and weaknesses of the use of novel plasmid-based standards are addressed in detail. In addition, the quantification system was designed to avoid the use of a reference gene (e.g., a single copy, species-specific gene) as normalizer, i.e., to perform a GMO quantification based on an absolute instead of a relative measurement. In fact, experimental evidences show that the use of reference genes adds variability to the measurement system because a second independent real-time PCR-based measurement must be performed. Moreover, for some reference genes no sufficient information on copy number in and among genomes of different lines is available, making adequate quantification difficult. Once developed, the method was subsequently validated according to IUPAC and ISO 5725 guidelines. Thirteen laboratories from 8 EU countries participated in the trial. Eleven laboratories provided results complying with the predefined study requirements. Repeatability (RSDr) values ranged from 8.7 to 15.9%, with a mean value of 12%. Reproducibility (RSDR) values ranged from 16.3 to 25.5%, with a mean value of 21%. Following Codex Alimentarius Committee guidelines, both the limits of detection and quantitation were determined to be <0.1%.

DIAGNOSIS AND QUANTIFICATION OF STRAWBERRY VEIN BANDING VIRUS USING MOLECULAR APPROACHES

PCR and gel analyses were used for detection and quantification of Strawberry vein banding virus. A viral DNA isolation procedure was optimized to allow quick isolation of total nucleic acids prior to PCR. Different aged leaves, petioles, flowers and roots from infected strawberry plants including a virus indicator and three commonly grown cultivars were used to determine the most suitable tissues for virus testing and evaluate different diagnostic procedures. Nucleic acid preparations were diluted 10-10,000 fold in sterile water to determine the detection limits of PCR and the relative virus titer of samples based on dilution end point assay. The highest virus titer was in old symptomatic leaves and the lowest was in petioles. However, virus titer varied greatly among source plants used. To determine the PCR inhibition limits, concentrated preparations (up to 100x higher than those used in routine diagnosis) were used as templates. False negative results were interpreted as inhibition of Taq DNA polymerase activity by host derived contaminants with the highest levels in terminal roots and the lowest in young terminal leaves. These data were used to design a framework in mass screening field samples by pooling multiple samples. Colorimetric PCR and dot blot hybridization assay were used to confirm the above findings.

A fluorescent based PCR assay for the detection and quantitation of Giardia duodenalis genotypes in mixed populations

A method based on the polymerase chain reaction has been developed for differentiating between genotypically and phenotypically distinct strains of Giardia duodenalis and quantifying the amount of initial template of the different genotypes in mixed populations. The assay relies on a sequence-specific probe, labelled with two fluorescent dyes, designed to bind within the small subunit ribosomal (SSU) RNA gene. This target region is amplified by primers specific for either Group 1 or Group 2-type isolates of G. duodenalis and the probe binds within the primer-targeted region. This quantitative method takes advantage of the 5′ nuclease activity of Taq DNA polymerase, which, on encountering a probe bound within the target DNA sequence cleaves it, causing it to become dissociated from the template. When the two fluorescent dyes bound to the probe are in close proximity (when the probe is intact), the interaction of the two dyes prevents the reporter dye from fluorescing. However, during the extension phase of amplification, the activity of the DNA polymerase causes the dyes to become separated and hence the reporter dye increases its fluorescent intensity. This release of fluorescence is directly related to the amplified amount of target template. This assay was developed with the aim of providing a unique method with which to investigate interactions within mixed populations of genetically distinct strains of G. duodenalis.

A simple method for elimination of false positive results in RT-PCR.

Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

ResonSense ®: simple linear fluorescent probes for quantitative homogeneous rapid polymerase chain reaction

Here we report a strand-specific fluorescent homogeneous assay format for rapid polymerase chain reaction (PCR). A number of similar assays are commonly used for research applications and are an ideal solution for a closed tube quantitative PCR. These assays use fluorescent resonant energy transfer (FRET) between donor and acceptor fluorescent moieties as the reporting mechanism. However, for different reasons these assays do not report amplification when very rapid cycling times are used. This is because current assays, such as TaqMan ®, are limited, in terms of assay speed, by the 5′–3′ exonuclease activity of Taq DNA polymerase. Other assays based on hybridisation require either a complex de-conformational event to occur, or require more than one probe to report amplification. Reducing the complexity of the experiment reduces costs in terms of design, optimisation and manufacture. Here, we describe ResonSense ® chemistries that use a simple linear fluorescent-labelled probe and a DNA minor-groove binding dye as either donor or acceptor moieties in a homogeneous assay format on the LightCycler ®. This assay format will provide for rapid analysis of samples and so it is particularly well suited to point-of-use testing.

Comparison between Taq DNA Polymerase and Its Stoffel Fragment for Quantitative Real-Time PCR with Hybridization Probes

In quantitative real-time PCR assays, fluorophor-labeled oligonucleotide probes are employed to generate sequence-specific signals for the quantitative evaluation. Whereas TaqMan probes have to be hydrolyzed during PCR by the endonucleolytic activity of Taq DNA polymerase to generate a signal, the hybridization probes in LightCycler assays must not be hydrolyzed. In this study, we demonstrate for four different targets that the probes are degraded during PCR by Taq DNA polymerase. Signal yield, quality of amplification curves, and accuracy of quantitative measurements can be improved using the Stoffel fragment lacking an endonucleolytic activity and TaqStart antibody suppressing the formation of nonspecific products, without laborious efforts to optimize the amplification protocol.

Fluorescence polarization in homogeneous nucleic acid analysis II: 5'-nuclease assay.

When the temperature and viscosity of the solvent is held constant, the degree of fluorescence polarization (FP) detected when a fluorescent dye is excited by plane polarized light depends mostly on the molecular weight of the dye molecule. By monitoring the FP of a fluorescent dye molecule, one can detect significant changes in the molecular weight of a fluorescent molecule without separation or purification. The 5'-nuclease (TaqMan) assay is a robust single nucleotide polymorphism genotyping method where an allele-specific probe that binds to a perfectly complementary target is cleaved by the 5'-nuclease activity of Taq DNA polymerase. Because the TaqMan probe is labeled with a fluorescent dye, it has high FP value when intact but a low FP value after cleavage. In this study, we compared the results of the 5'-nuclease assay based on standard fluorescence intensity readings and FP readings when genotyping 90 individuals with 20 single nucleotide polymorphisms. Our results show that FP is just as robust and reliable as the standard fluorescence detection method. Use of FP detection makes it possible to reduce the cost of TaqMan probes by abrogating the need for a fluorescence quencher.

Detection of Ralstonia solanacearum from potato tissue by post‐enrichment TaqMan PCR*

TaqMan polymerase chain reaction (PCR) exploits the 5′‐nuclease activity of Taq DNA polymerase in conjunction with fluorogenic DNA probes. A positive PCR reaction results in a measurable emission of fluorescence within 2 h. TaqMan assays for several plant‐pathogenic bacteria have been designed, including the potato brown rot pathogen Ralstonia solanacearum. Enrichment techniques designed to improve the detection of low pathogen populations in potato tissue are also suitable for use with TaqMan PCR. The quantitative aspect of TaqMan PCR enables relative population level differences between pre‐enriched and enriched samples to be demonstrated and thus cell viability to be evaluated.

Real-time Quantitative PCR for Retrovirus-like Particle Quantification in CHO Cell Culture

Chinese hamster ovary (CHO) cells have been widely used to manufacture recombinant proteins intended for human therapeutic uses. Retrovirus-like particles, which are apparently defective and non-infectious, have been detected in all CHO cells by electron microscopy (EM). To assure viral safety of CHO cell-derived biologicals, quantification of retrovirus-like particles in production cell culture and demonstration of sufficient elimination of such retrovirus-like particles by the down-stream purification process are required for product market registration worldwide. EM, with a detection limit of 1×106particles/ml, is the standard retrovirus-like particle quantification method. The whole process, which requires a large amount of sample (3–6 litres), is labour intensive, time consuming, expensive, and subject to significant assay variability. In this paper, a novel real-time quantitative PCR assay (TaqMan assay) has been developed for the quantification of retrovirus-like particles. Each retrovirus particle contains two copies of the viral genomic particle RNA (pRNA) molecule. Therefore, quantification of retrovirus particles can be achieved by quantifying the pRNA copy number, i.e. every two copies of retroviral pRNA is equivalent to one retrovirus-like particle. The TaqMan assay takes advantage of the 5′→3′ exonuclease activity of Taq DNA polymerase and utilizes the PRISM™ 7700 Sequence Detection System of PE Applied Biosystems (Foster City, CA, U.S.A.) for automated pRNA quantification through a dual-labelled fluorogenic probe. The TaqMan quantification technique is highly comparable to the EM analysis. In addition, it offers significant advantages over the EM analysis, such as a higher sensitivity of less than 600particles/ml, greater accuracy and reliability, higher sample throughput, more flexibility and lower cost. Therefore, the TaqMan assay should be used as a substitute for EM analysis for retrovirus-like particle quantification in CHO cell-based production system.

Detection of Ralstonia solanacearum strains with a quantitative, multiplex, real-time, fluorogenic PCR (TaqMan) assay.

A fluorogenic (TaqMan) PCR assay was developed to detect Ralstonia solanacearum strains. Two fluorogenic probes were utilized in a multiplex reaction; one broad-range probe (RS) detected all biovars of R. solanacearum, and a second more specific probe (B2) detected only biovar 2A. Amplification of the target was measured by the 5' nuclease activity of Taq DNA polymerase on each probe, resulting in emission of fluorescence. TaqMan PCR was performed with DNA extracted from 42 R. solanacearum and genetically or serologically related strains to demonstrate the specificity of the assay. In pure cultures, detection of R. solanacearum to >/=10(2) cells ml(-1) was achieved. Sensitivity decreased when TaqMan PCR was performed with inoculated potato tissue extracts, prepared by currently recommended extraction procedures. A third fluorogenic probe (COX), designed with the potato cytochrome oxidase gene sequence, was also developed for use as an internal PCR control and was shown to detect potato DNA in an RS-COX multiplex TaqMan PCR with infected potato tissue. The specificity and sensitivity of the assay, combined with high speed, robustness, reliability, and the possibility of automating the technique, offer potential advantages in routine indexing of potato tubers and other plant material for the presence of R. solanacearum.

An improved method of DNA isolation from plants collected in the field and conserved in saturated NaCl/CTAB solution

SummaryŠtorchová, H., Hrdličková, R., Chrtek, J., Jr., Tetera, M., Fitze, D. &amp; Fehrer, J.: An improved method of DNA isolation from plants collected in the field and conserved in saturated NaCl/CTAB solution. – Taxon 49: 79‐84. 2000. – ISSN 0040‐0262.A simple method for isolation of genomic DNA from wild plants sampled in remote field areas is presented. The protocol combines NaCl/CTAB leaf preservation with sorbitol extraction of secondary compounds which often contain inhibitors of Taq DNA polymerase activity. The obtained DNA is suitable for random amplified polymorphic DNA (RAPD) analysis of plant populations as well as for specific amplification of chloroplast DNA sequences. The NaCl/CTAB leaf preservation is a powerful alternative to silica gel drying‐based preservation.

High‐resolution typing for HLA‐DRB1*15 and ‐DRB1*16 by fluorescence‐marked sequence‐specific priming (TaqMan assay)

Sequence-specific primed polymerase chain reaction (PCR-SSP) is widely used in HLA laboratories. The TaqMan method, which is described here for high-resolution typing of HLA-DRB1*15 and -DRB1*16, does not require elaborate and time-consuming post-PCR detection steps. In this one-tube assay, conventional PCR-SSP and fluorescence detection of the amplicon with a doubly labeled fluorescent probe are combined: a fluorogenic hybridization probe (FHP) labeled with a spectral resolvable fluorescent reporter dye (FAM or TET) at its 5' terminus and a common quencher dye (TAMRA) at its 3' terminus is cleaved by the 5' nuclease activity of Taq DNA polymerase only if the target sequence is amplified. An increase of fluorescence intensity indicates a successful amplification. For high-resolution typing of HLA-DRB1*15 and -DRB1*16 alleles we designed two FHPs and 14 specific primer mixes (7 for DR15 and 7 for DR16). Amplification of the specific sequence was detected by a FAM-labeled FHP, whereas amplification of the internal control was detected by a TET-labeled FHP. We were able to type all heterozygous DRB1*15/DRB1*16 subtype combinations. For evaluation, 60 HLA-DRB1*15-positive and 40 HLA-DRB1*16-positive individuals were typed and the results were compared with conventional PCR-SSP DR15/16 subtyping. There were no discrepancies between the two methods. The TaqMan method is an alternative to conventional PCR-SSP typing which is suitable for routine use in HLA laboratories.

Two sensitive PCR-based methods for detection of hepatitis B virus variants associated with reduced susceptibility to lamivudine.

Two novel assays, a restriction fragment length polymorphism (RFLP) assay and an assay based on the 5'-nuclease activity of Taq DNA polymerase, were developed for screening viral variants in lamivudine-treated patients' sera containing <1,000 copies of the hepatitis B virus (HBV) genome per ml. Both assays were designed to detect single-nucleotide changes within the HBV DNA polymerase gene that are associated with lamivudine resistance in vitro and have been used to screen a number of patients' sera for variant virus. Results obtained with these assays and standard sequencing technology were compared with regard to throughput, ability to detect individual virus species present at low concentrations, and ability to detect, distinguish, and quantitate wild-type (wt) and HBV tyrosine methionine(552) aspartate aspartate motif variants in mixed viral populations. Unlike DNA sequencing, both assays are amenable to high-throughput screening and were shown to be able to quantitatively detect variant virus in the presence of a background of wt virus. As with DNA sequencing, both new assays incorporate a PCR amplification step and are able to detect the relatively low amounts of virus found in lamivudine-treated patients' sera. However, these assays are far less labor intensive than the DNA-sequencing techniques presently in use. Overall, the RFLP assay was more sensitive than DNA sequencing in detecting and determining the ratios of wt to variant virus. Furthermore, the RFLP assay and 5'-nuclease assay were equally sensitive in the detection of mixed viral species, but the RFLP assay was superior to the 5'-nuclease assay in the quantitation of mixed viral species. These assays should prove useful for further understanding of virological response to therapy and disease progression.

Real Time Quantitative PCR as a Method to Evaluate Simian Virus 40 Removal During Pharmaceutical Protein Purification

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5′-3′ exonuclease activity of Taq DNA polymerase and utilizes the PRISMTM7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.

A Real Time Quantitative PCR-based Method for the Detection and Quantification of Simian Virus 40

A real time quantitative PCR-based simian virus 40 (SV40) detection and quantification method has been developed. This method takes advantage of the 5′ to 3′-exonuclease activity of Taq DNA polymerase and utilizes the PRISMTM7700 sequence detection system of PE Applied Biosystems for direct monitoring of PCR product accumulation through a dual-labelled fluorogenic probe. This method provides accurate, precise and reproducible quantification of SV40 DNA over a linear dynamic range of at least 100000-fold with a minimum detection level of 6·4 copy equivalents/μL of SV40 viral particle in test samples. The sample preparation procedure employed allows for efficient and consistent recovery of SV40 DNA from test samples. High concentrations of protein and cellular DNA presenting in test samples have been demonstrated to have no impact on SV40 quantification. This method offers significant advantages over other PCR methods and cell-based infectivity assays currently available for SV40 detection and quantification. The availability of this method should greatly facilitate the pathogenic investigation of SV40, as well as viral clearance evaluations required for the development of new biological products.