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Zip nucleic acid: a new reliable method to increase the melting temperature of real-time PCR probes.

ABSTRACT: TaqMan genotyping with real-time PCR is a reliable method for single nucleotide polymorphism detection, which is done by probes. These oligonucleotides should be short enough to avoid mismatch hybridization, as well as having 5-10°C higher melting temperature than the primers of real-time PCR reaction. One approach for these qualities is to conjugate the probe with minor groove binder (MGB). Having no access to MGB probes, we searched for an alternative. In the current study, we used Zip Nucleic Acids (ZNA) as probes to increase its stability and melting temperature. Our aim was to genotype the -265 T/C changes of Apolipoprotein A-2 gene. We set up the real-time PCR reaction with ZNA probes, and by repeating the reactions, we confirmed the reliability of this new approach. It is now recommended to use ZNA probes, as an alternative to MGB probes, to increase the probe Tm value and its binding to target DNA.

SUBMITTER: Alvandi E

PROVIDER: S-EPMC3922743 | biostudies-literature | 2014 Feb

REPOSITORIES: biostudies-literature

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Zip nucleic acid: a new reliable method to increase the melting temperature of real-time PCR probes.

Alvandi Ehsan E Koohdani Fariba F

Journal of diabetes and metabolic disorders 20140204 1

TaqMan genotyping with real-time PCR is a reliable method for single nucleotide polymorphism detection, which is done by probes. These oligonucleotides should be short enough to avoid mismatch hybridization, as well as having 5-10°C higher melting temperature than the primers of real-time PCR reaction. One approach for these qualities is to conjugate the probe with minor groove binder (MGB). Having no access to MGB probes, we searched for an alternative. In the current study, we used Zip Nucleic ...[more]

PMID: 24495816

Similar Datasets

Zip nucleic acids are potent hydrolysis probes for quantitative PCR.

Project description:Zip nucleic acids (ZNAs) are oligonucleotides conjugated with cationic spermine units that increase affinity for their target. ZNAs were recently shown to enable specific and sensitive reactions when used as primers for polymerase chain reaction (PCR) and reverse-transcription. Here, we report their use as quantitative PCR hydrolysis probes. Ultraviolet duplex melting data demonstrate that attachment of cationic residues to the 3' end of an oligonucleotide does not alter its ability to discriminate nucleotides nor the destabilization pattern relative to mismatch location in the oligonucleotide sequence. The stability increase provided by the cationic charges allows the use of short dual-labeled probes that significantly improve single-nucleotide polymorphism genotyping. Longer ZNA probes were shown to display reduced background fluorescence, therefore, generating greater sensitivity and signal level as compared to standard probes. ZNA probes thus provide broad flexibility in assay design and also represent an effective alternative to minor groove binder- and locked nucleic-acid-containing probes.

| S-EPMC2853127 | biostudies-literature

Asymmetric real-time PCR and multiplex melting curve analysis with TaqMan probes for detecting PIK3CA mutations.

Project description:The data in this article are related to the research article entitled "Optimization of melting analysis with TaqMan probes for detection of KRAS, NRAS, and BRAF mutations" Botezatu et al. [1]. Somatic mutations in the PIK3CA gene ("hot spots" in exons 9 and 20) are found in many human cancers, and their presence can determine prognosis and a treatment strategy. An effective method of mutation scanning PIK3CA in clinical laboratories is DNA Melting Analysis (DMA) (Vorkas et al., 2010; Simi et al., 2008) [2], [3]. It was demonstrated recently that the TaqMan probes which have been long used in Real Time PCR may also be utilized in DMA (Huang et al., 2011) [4]. After optimization of this method Botezatu et al. [1], it was used for multiplex scanning PIK3CA hotspot mutations in formalin-fixed paraffin-embedded (FFPE) samples from patients with colorectal and lung cancer.

| S-EPMC4669479 | biostudies-literature

Real-time PCR and melting curve analysis for reliable and rapid detection of SHV extended-spectrum beta-lactamases.

Project description:Extended-spectrum beta-lactamases (ESBLs), e.g., ESBLs of the TEM or SHV type, compromise the efficacies of expanded-spectrum cephalosporins. An SHV non-ESBL that hydrolyzes only narrow-spectrum cephalosporins can be converted into an SHV ESBL through substitutions at three amino acid positions, 179, 238, or 238--240. In order to improve detection of SHV ESBLs, a novel method, based on real-time PCR monitored with fluorescently labeled hybridization probes and followed by melting curve analysis, was developed. It is able to (i) detect bla(SHV) genes with high degrees of sensitivity and specificity, (ii) discriminate between bla(SHV non-ESBL) and bla(SHV ESBL), and (iii) categorize the SHV ESBL producers into three phenotypically relevant subgroups. This method, termed the SHV melting curve mutation detection method, represents a powerful tool for epidemiological studies with SHV ESBLs. It even has the potential to be used in the diagnostic microbiology laboratory, because up to 32 clinical isolates can be processed in less than 1 h by starting with just a few bacterial colonies.

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Real-time PCR genotyping using displacing probes.

Project description:Simple and reliable genotyping technology is a key to success for high-throughput genetic screening in the post-genome era. Here we have developed a new real-time PCR genotyping approach that uses displacement hybridization-based probes: displacing probes. The specificity of displacing probes could be simply assessed through denaturation analysis before genotyping was implemented, and the probes designed with maximal specificity also showed the greatest detection sensitivity. The ease in design, the simple single-dye labeling chemistry and the capability to adopt degenerated negative strands for point mutation genotyping make the displacing probes both cost effective and easy to use. The feasibility of this method was first tested by detecting the C282Y mutation in the human hemochromatosis gene. The robustness of this approach was then validated by simultaneous genotyping of five different types of mutation in the human beta-globin gene. Sixty-two human genomic DNA samples with nine known genotypes were accurately detected, 32 random clinical samples were successfully screened and 114 double-blind DNA samples were all correctly genotyped. The combined merits of reliability, flexibility and simplicity should make this method suitable for routine clinical testing and large-scale genetic screening.

| S-EPMC407835 | biostudies-other

Assessment for Melting Temperature Measurement of Nucleic Acid by HRM.

Project description:High resolution melting (HRM), with a high sensitivity to distinguish the nucleic acid species with small variations, has been widely applied in the mutation scanning, methylation analysis, and genotyping. For the aim of extending HRM for the evaluation of thermal stability of nucleic acid secondary structures on sequence dependence, we investigated effects of the dye of EvaGreen, metal ions, and impurities (such as dNTPs) on melting temperature (Tm ) measurement by HRM. The accuracy of HRM was assessed as compared with UV melting method, and little difference between the two methods was found when the DNA Tm was higher than 40°C. Both insufficiency and excessiveness of EvaGreen were found to give rise to a little bit higher Tm , showing that the proportion of dye should be considered for precise Tm measurement of nucleic acids. Finally, HRM method was also successfully used to measure Tm s of DNA triplex, hairpin, and RNA duplex. In conclusion, HRM can be applied in the evaluation of thermal stability of nucleic acid (DNA or RNA) or secondary structural elements (even when dNTPs are present).

| S-EPMC5090098 | biostudies-literature

A novel universal real-time PCR system using the attached universal duplex probes for quantitative analysis of nucleic acids.

Project description:BackgroundReal-time PCR techniques are being widely used for nucleic acids analysis, but one limitation of current frequently employed real-time PCR is the high cost of the labeled probe for each target molecule.ResultsWe describe a real-time PCR technique employing attached universal duplex probes (AUDP), which has the advantage of generating fluorescence by probe hydrolysis and strand displacement over current real-time PCR methods. AUDP involves one set of universal duplex probes in which the 5' end of the fluorescent probe (FP) and a complementary quenching probe (QP) lie in close proximity so that fluorescence can be quenched. The PCR primer pair with attached universal template (UT) and the FP are identical to the UT sequence. We have shown that the AUDP technique can be used for detecting multiple target DNA sequences in both simplex and duplex real-time PCR assays for gene expression analysis, genotype identification, and genetically modified organism (GMO) quantification with comparable sensitivity, reproducibility, and repeatability with other real-time PCR methods.ConclusionThe results from GMO quantification, gene expression analysis, genotype identification, and GMO quantification using AUDP real-time PCR assays indicate that the AUDP real-time PCR technique has been successfully applied in nucleic acids analysis, and the developed AUDP real-time PCR technique will offer an alternative way for nucleic acid analysis with high efficiency, reliability, and flexibility at low cost.

| S-EPMC2443376 | biostudies-literature

An Inexpensive and Accurate Reverse Transcription-PCR-Melting Temperature Analysis Assay for Real-Time Influenza Virus B Lineage Discrimination.

Project description:In this work, we describe a SYBR-Green one-step reverse transcription-PCR protocol coupled with a melting temperature analysis (RT-PCR-Tm ), which allows the discrimination of influenza B lineages Yamagata and Victoria. The assay is performed using a regular real-time thermocycler and is based on differences in melting temperature (Tm ) of a 131-bp amplicon, obtained from a conserved region of hemagglutinin gene. A total of 410 samples collected during the 2004, 2008, and 2010-2017 influenza seasons in Brazil were tested, and the lineages were correctly characterized using their melting profiles. The temperature range is significantly different between both lineages throughout the time (Mann-Whitney test; P < 0.0001, confidence interval = 95%), and the Tm is not affected by viral load (Spearman correlation test; r?=?0.287, P = 2.245?×?10-9). The simplicity and cost-effectiveness of this protocol make it an option for influenza B lineage surveillance worldwide.

| S-EPMC6879292 | biostudies-literature

Zip Nucleic Acids: new high affinity oligonucleotides as potent primers for PCR and reverse transcription.

Project description:Most nucleic acid-based technologies rely upon sequence recognition between an oligonucleotide and its nucleic acid target. With the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, novel modified oligonucleotides named Zip nucleic acids (ZNAs) were recently developed. ZNAs are oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotide. It was demonstrated that the melting temperature of a hybridized ZNA is easily predictable and increases linearly with the length of the oligocation. Furthermore, ZNAs retain the ability to discriminate between a perfect match and a single base-pair-mismatched complementary sequence. Using quantitative PCR, we show here that ZNAs are specific and efficient primers displaying an outstanding affinity toward their genomic target. ZNAs are particularly efficient at low magnesium concentration, low primer concentrations and high annealing temperatures, allowing to improve the amplification in AT-rich sequences and potentially multiplex PCR applications. In reverse transcription experiments, ZNA gene-specific primers improve the yield of cDNA synthesis, thus increasing the accuracy of detection, especially for genes expressed at low levels. Our data suggest that ZNAs exhibit faster binding kinetics than standard and locked nucleic acid-containing primers, which could explain why their target recognition is better for rare targets.

| S-EPMC2770653 | biostudies-literature

Comparison of viremia of type II porcine reproductive and respiratory syndrome virus in naturally infected pigs by zip nucleic acid probe-based real-time PCR.

Project description:BACKGROUND: Porcine reproductive and respiratory syndrome virus (PRRSV) is a RNA virus with high genetic variation. This virus causes significant economic losses in most pig-producing countries. The clinical presentation of PRRSV ranges from asymptomatic to devastating. In this study, we developed a sensitive and specific zip nucleic acid probe-based real-time PCR assay to evaluate the viremia of natural PRRSV-infected pigs in Taiwan. Serum samples were collected from 577 pigs aged 5-12 weeks. These include 444 clinically healthy pigs and 133 symptomatic pigs were confirmed to have porcine respiratory disease complex (PRDC). RESULTS: Viremia was quantified in 79 of the 444 (17.8%) clinically healthy pigs and in 112 of the 133 (84.2%) PRDC cases. Viremias were significantly more common in pigs with PRDC compared with the clinically healthy pigs (P <0.0001). These results suggest that a high viral load is a major feature of PRRSV-affected pigs. CONCLUSIONS: ZNA probe-based real-time PCR can be a useful tool to diagnose symptomatic and asymptomatic PRRSV-infected pigs. The presence of this marker in a sample of animals with high PRRSV loads (>10(4.2) PRRSV genomes/?l of serum) seems to indicate that it correlates with the presence of PRDC in pigs.

| S-EPMC3847877 | biostudies-literature

Modified low cost SNP genotyping technique using cycle threshold (Ct) & melting temperature (Tm) values in allele specific real-time PCR.

Project description:Genotyping has now become one of the major diagnostic means for almost all diseases. Among the advanced techniques that are used to study single nucleotide polymorphisms (SNPs), only a few are applicable for routine disease diagnosis. Their applicability mainly depends on three factors: cost, time, and accuracy. The primary objective of this study was to propose allele-specific real-time PCR as a rapid, low cost and simple genotyping method for routine diagnostics.Two SNPs, rs3014866 and rs2149356 were analysed using allele-specific real-time PCR. The polymerase chain reaction was carried out using RealQ PCR master mix containing SYBR Green DNA I dye followed by melt curve analysis. The results were validated by agarose gel electrophoresis and DNA sequencing.The allelic discrimination and zygosity of the two SNPs were assessed by combined cycle threshold (Ct) and melting temperature (T m ) values. Variations in Ct and T m values among the two alleles were observed in both rs3014866 (Ct: C allele - 24 ± 1, T allele - 27 ± 1; T m: C allele - 82.5 ± 0.3, T allele - 86.3 ± 0.2) and rs2149356 (Ct: C allele - 24 ± 1, A allele - 26 ± 1; T m: C allele - 79.4 ± 0.2, A allele - 80.4 ± 0.3). Based on the variations, homozygous and heterozygous alleles were detect ed. Agarose gel electrophoresis and DNA sequencing also confirmed the allelic variation and zygosity observed in real-time PCR.In diagnostic settings where a large number of samples are analysed daily, allele-specific real-time PCR assay may serve as a simple, low cost and efficient method of genotyping.

| S-EPMC4743342 | biostudies-literature

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