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.     

Simultaneous mutation scanning and genotyping by high-resolution DNA melting analysis

This protocol permits the simultaneous mutation scanning and genotyping of PCR products by high-resolution DNA melting analysis. This is achieved using asymmetric PCR performed in the presence of a saturating fluorescent DNA dye and unlabeled oligonucleotide probes. Fluorescent melting curves of both PCR amplicons and amplicon-probe duplexes are analyzed. The shape of the PCR amplicon melting transition reveals the presence of heterozygotes, whereas specific genotyping is enabled by melting of the unlabeled probe-amplicon duplex. Unbiased hierarchal clustering of melting transitions automatically groups different sequence variants; this allows common variants to be easily recognized and genotyped. This technique may be used in both laboratory research and clinical settings to study single-nucleotide polymorphisms and small insertions and deletions, and to diagnose associated genetic disorders. High-resolution melting analysis accomplishes simultaneous gene scanning and mutation genotyping in a fraction of the time required when using traditional methods, while maintaining a closed-tube environment. The PCR requires <30 min (capillaries) or 1.5 h (96- or 384-well plates) and melting acquisition takes 1-2 min per capillary or 5 min per plate.     

Real-time multiplex PCR assays

The ability to multiplex PCR by probe color and melting temperature (T(m)) greatly expands the power of real-time analysis. Simple hybridization probes with only a single fluorescent dye can be used for quantification and allele typing. Different probes are labeled with dyes that have unique emission spectra. Spectral data are collected with discrete optics or dispersed onto an array for detection. Spectral overlap between dyes is corrected by using pure dye spectra to deconvolute the experimental data by matrix algebra. Since fluorescence is temperature dependent and depends on the dye, spectral overlap and color compensation constants are also temperature dependent. Single-labeled probes are easier to synthesize and purify than more complex probes with two or more dyes. In addition, the fluorescence of single-labeled probes is reversible and depends only on hybridization of the probe to the target, allowing study of the melting characteristics of the probe. Although melting curves can be obtained during PCR, data are usually acquired at near-equilibrium rates of 0.05-0.2 degrees C/s after PCR is complete. Using rapid-cycle PCR, amplification requires about 20 min followed by a 10-min melting curve, greatly reducing result turnaround time. In addition to dye color, melting temperature can be used for a second dimension of multiplexing. Multiplexing by color and T(m) creates a "virtual" two-dimensional multiplexing array without the need for an immobilized matrix of probes. Instead of physical separation along the X and Y axes, amplification products are identified by different fluorescence spectra and melting characteristics.     

Eprobe Mediated Real-Time PCR Monitoring and Melting Curve Analysis

Real-time monitoring of PCR is one of the most important methods for DNA and RNA detection widely used in research and medical diagnostics. Here we describe a new approach for combined real-time PCR monitoring and melting curve analysis using a 3′ end-blocked Exciton-Controlled Hybridization-sensitive fluorescent Oligonucleotide (ECHO) called Eprobe. Eprobes contain two dye moieties attached to the same nucleotide and their fluorescent signal is strongly suppressed as single-stranded oligonucleotides by an excitonic interaction between the dyes. Upon hybridization to a complementary DNA strand, the dyes are separated and intercalate into the double-strand leading to strong fluorescence signals. Intercalation of dyes can further stabilize the DNA/DNA hybrid and increase the melting temperature compared to standard DNA oligonucleotides. Eprobes allow for specific real-time monitoring of amplification reactions by hybridizing to the amplicon in a sequence-dependent manner. Similarly, Eprobes allow for analysis of reaction products by melting curve analysis. The function of different Eprobes was studied using the L858R mutation in the human epidermal growth factor receptor (EGFR) gene, and multiplex detection was demonstrated for the human EGFR and KRAS genes using Eprobes with two different dyes. Combining amplification and melting curve analysis in a single-tube reaction provides powerful means for new mutation detection assays. Functioning as “sequence-specific dyes”, Eprobes hold great promises for future applications not only in PCR but also as hybridization probes in other applications.     

Closed-tube genotyping of apolipoprotein E by isolated-probe PCR with multiple unlabeled probes and high-resolution DNA melting analysis

Isolated-probe PCR (IP-PCR) is a method that combines asymmetric PCR, unlabeled probes, and high-resolution DNA melting while maintaining a closed tube system. A double-stranded DNA (dsDNA) dye LCGreen I was used to detect the unlabeled probes. LCGreen I is also used to detect the 277-base pair PCR product peak as an internal amplification control. To accomplish this, IP-PCR separates the asymmetric PCR amplification step and the detection step of the unlabeled probes. This prevents the probes from interfering with the amplification of the DNA target. The samples are then melted using a high-resolution DNA melting instrument: the HR-1. The closed tube system virtually eliminates PCR product contamination or sample carryover The target apolipoprotein E (APOE) was chosen to test the IP-PCR technique. APOE contains two single nucleotide polymorphisms (SNPs) located 139 base pairs apart in a GC-rich region of the human genome. The results from this study show that the IP-PCR technique was able to determine the correct APOE genotype for each of the 101 samples. The IP-PCR technique should also be useful in detecting SNPs in other high-GC regions of the human genome.     

Comparing mono- and divalent DNA groove binding cyanine dyes -- binding geometries, dissociation rates, and fluorescence properties

The unsymmetrical cyanine dyes BOXTO-PRO and BOXTO-MEE were derived from the DNA groove binder BOXTO, by adding a positively charged or a non-ionic hydrophilic tail to BOXTO, respectively. The main objective was to obtain more efficient DNA probes, for instance in electrophoresis and microscopy, by slowing down the dissociation of BOXTO from DNA. The interactions with mixed sequence DNA was studied with fluorescence and absorbance spectroscopy, stopped-flow dissociation and gel electrophoresis. Both the derivatives are groove bound as BOXTO, and have similar fluorescence properties when bound to mixed sequence DNA in free solution. BOXTO-PRO exhibits a slower dissociation than BOXTO from DNA, whereas the dissociation rate for BOXTO-MEE is faster and, unexpectedly independent of the ionic strength. During gel electrophoresis both BOXTO-PRO and BOXTO-MEE exhibit a faster dissociation rate than BOXTO. Still, BOXTO-PRO seems to be a good alternative as DNA probe, especially for applications in free solution where the dissociation is slower than for the corresponding intercalator TOPRO-1.     

Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis

Following the initial report of the use of SYBR Green I for real-time polymerase chain reaction (PCR) in 1997, little attention has been given to the development of alternative intercalating dyes for this application. This is surprising considering the reported limitations of SYBR Green I, which include limited dye stability, dye-dependent PCR inhibition, and selective detection of amplicons during DNA melting curve analysis of multiplex PCRs. We have tested an alternative to SYBR Green I and report the first detailed evaluation of the intercalating dye SYTO9. Our findings demonstrate that SYTO9 produces highly reproducible DNA melting curves over a broader range of dye concentrations than does SYBR Green I, is far less inhibitory to PCR than SYBR Green I, and does not appear to selectively detect particular amplicons. The low inhibition and high melting curve reproducibility of SYTO9 means that it can be readily incorporated into a conventional PCR at a broad range of concentrations, allowing closed tube analysis by DNA melting curve analysis. These features simplify the use of intercalating dyes in real-time PCR and the improved reproducibility of DNA melting curve analysis will make SYTO9 useful in a diagnostic context.     

Combination probes with intercalating anchors and proximal fluorophores for DNA and RNA detection

A new class of modified oligonucleotides (combination probes) has been designed and synthesised for use in genetic analysis and RNA detection. Their chemical structure combines an intercalating anchor with a reporter fluorophore on the same thymine nucleobase. The intercalator (thiazole orange or benzothiazole orange) provides an anchor, which upon hybridisation of the probe to its target becomes fluorescent and simultaneously stabilizes the duplex. The anchor is able to communicate via FRET to a proximal reporter dye (e.g. ROX, HEX, ATTO647N, FAM) whose fluorescence signal can be monitored on a range of analytical devices. Direct excitation of the reporter dye provides an alternative signalling mechanism. In both signalling modes, fluorescence in the unhybridised probe is switched off by collisional quenching between adjacent intercalator and reporter dyes. Single nucleotide polymorphisms in DNA and RNA targets are identified by differences in the duplex melting temperature, and the use of short hybridization probes, made possible by the stabilisation provided by the intercalator, enhances mismatch discrimination. Unlike other fluorogenic probe systems, placing the fluorophore and quencher on the same nucleobase facilitates the design of short probes containing multiple modifications. The ability to detect both DNA and RNA sequences suggests applications in cellular imaging and diagnostics.     

Oligonucleotide derivatives in the nucleic acid hybridization analysis. II. Isothermal signal amplification in process of DNA analysis by minisequencing

The isothermal amplification of reporter signal via limited probe extension (minisequencing) upon hybridization of nucleic acids has been studied. The intensity of reporter signal has been shown to increase due to enzymatic labeling of multiple probes upon consecutive hybridization with one DNA template both in homophase and heterophase assays using various kinds of detection signal: radioisotope label, fluorescent label, and enzyme-linked assay. The kinetic scheme of the process has been proposed and kinetic parameters for each step have been determined. The signal intensity has been shown to correlate with physicochemical characteristics of both complexes: probe/DNA and product/DNA. The maximum intensity has been observed at minimal difference between the thermodynamic stability of these complexes, provided the reaction temperature has been adjusted near their melting temperature values; rising or lowering the reaction temperature reduces the amount of reporting product. The signal intensity has been shown to decrease significantly upon hybridization with the DNA template containing single-nucleotide mismatches. Limited probe extension assay is useful not only for detection of DNA template but also for its quantitative characterization.     

Monitoring of fluorescence during DNA melting as a method for discrimination and detection of PCR products in variety identification

DNA melting curves of genotype-specific PCR fragments were used to differentiate between species and amongst varieties of cereals. Melting curves were generated by ramping the temperature of PCR fragments through their dissociation temperature in the presence of a double-stranded DNA binding dye. Genotypes were discriminated by differences in the position and shape of the melting curve which is a function of the fragment's sequence, length and GC content. Amplification of 5S ribosomal RNA genes generated species-specific fragments for six of the major cereal crops. Of the 15 possible pairwise comparisons, 13 distinctions could be reliably made using melting curve position data. Wheat varieties were identified by the melting profiles of PCR products generated using microsatellite primers. DNA melting curve analysis was conveniently coupled with capillary-PCR using a LightCycler instrument to provide a rapid method of genotyping in cereals.     

Comparison of membranes and glass slides for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes

Analyses of complex microbial communities are becoming increasingly important. Bottlenecks in these analyses, however, are the tools to actually describe the biodiversity. Novel protocols for a DNA array based analyzes of microbial communities are presented. In these protocols, the specificity obtained by sequence-specific labeling of DNA probes is combined with the possibility of detecting several different probes simultaneously by DNA array hybridization. The gene encoding 16S ribosomal RNA was chosen as the target in these analyses. This gene contains both universally conserved regions, and regions with relatively high variability. The universally conserved regions are used for PCR amplification primers, while the variable regions are used for the specific probes. Arrays prepared on positively charged nylon membranes and coated glass slides were compared. The advantage of using membranes is that chromogenic signal amplification can be used for the detection. Furthermore, the chromogenic detection does not require any sophisticated equipment. The advantage of the glass slides is that multiple fluorescence colors can be detected simultaneously, and that internal controls can be used for normalization. This approach is also suited for high throughput screenings.     

Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping

High-throughput methods for assaying DNA variation require two important steps: (i) discriminating the variation and (ii) detecting the signal. In this report, we describe a novel SNP genotyping method that we refer to as melting curve analysis of SNPs (McSNP). McSNP combines a classic approach for discriminating alleles, restriction enzyme digestion, with a more recent method for detecting DNA fragments, melting curve analysis. Melting curve analysis is performed by slowly heating DNA fragments in the presence of the dsDNA-specific fluorescent dye SYBR Green I. As the sample is heated, fluorescence rapidly decreases when the melting temperature of a particular fragment is reached. We show that it is possible to determine the composition of simple mixtures of DNA fragments, such as those that result from restriction enzyme digestions of short PCR products. McSNP is well suited for high-throughput genotyping because 96 samples can be analyzed and automatically scored in 20 min. Our results clearly demonstrate that McSNP is a simple, inexpensive, and accurate means of genotyping SNP variation.     

Quantitative PCR in the diagnosis of Leishmania

Polymerase chain reaction (PCR) is a sensitive and rapid method for the diagnosis of canine Leishmania infection and can be performed on a variety of biological samples, including peripheral blood, lymph node, bone marrow and skin. Standard PCR requires electrophoretic analysis of the amplification products and is usually not suitable for quantification of the template DNA (unless competitor-based or other methods are developed), being of reduced usefulness when accurate monitoring of target DNA is required. Quantitative real-time PCR allows the continuous monitoring of the accumulation of PCR products during the amplification reaction. This allows the identification of the cycle of near-logarithmic PCR product generation (threshold cycle) and, by inference, the relative quantification of the template DNA present at the start of the reaction. Since the amplification product are monitored in "real-time" as they form cycle-by-cycle, no post-amplification handling is required. The absolute quantification is performed according either to an internal standard co-amplified with the sample DNA, or to an external standard curve obtained by parallel amplification of serial known concentrations of a reference DNA sequence. From the quantification of the template DNA, an estimation of the relative load of parasites in the different samples can be obtained. The advantages compared to standard and semi-quantitative PCR techniques are reduction of the assay's time and contamination risks, and improved sensitivity. As for standard PCR, the minimal components of the quantitative PCR reaction mixture are the DNA target of the amplification, an oligonucleotide primer pair flanking the target sequence, a suitable DNA polymerase, deoxynucleotides, buffer and salts. Different technologies have been set up for the monitoring of amplification products, generally based on the use of fluorescent probes. For instance, SYBR Green technology is a non-specific detection system based on a fluorescent dsDNA intercalator and it is applicable to all potential targets. TaqMan technology is more specific since performs the direct assessment of the amount of amplified DNA using a fluorescent probe specific for the target sequence flanked by the primer pair. This probe is an oligonucleotide labelled with a reporter dye (fluorescent) and a quencher (which absorbs the fluorescent signal generated by the reporter). The thermic protocol of amplification allows the binding of the fluorescent probe to the target sequence before the binding of the primers and the starting of the polymerization by Taq polymerase. During polymerization, 5'-3' exonuclease activity of Taq polymerase digests the probe and in this way the reporter dye is released from the probe and a fluorescent signal is detected. The intensity of the signal accumulates at the end of each cycle and is related to the amount of the amplification product. In recent years, quantitative PCR methods based either on SYBR Green or TaqMan technology have been set up for the quantification of Leishmania in mouse liver, mouse skin and human peripheral blood, targeting either single-copy chromosomal or multi-copy minicircle sequences with high sensitivity and reproducibility. In particular, real-time PCR seems to be a reliable, rapid and noninvasive method for the diagnosis and follow up of visceral leishmaniasis in humans. At present, the application of real-time PCR for research and clinical diagnosis of Leishmania infection in dogs is still foreseable. As for standard PCR, the high sensitivity of real-time PCR could allow the use of blood sampling that is less invasive and easily performed for monitoring the status of the dogs. The development of a real-time PCR assay for Leishmania infantum infection in dogs could support the standard and optimized serological and PCR methods currenly in use for the diagnosis and follow-up of canine leishmaniasis, and perhaps prediction of recurrences associated with tissue loads of residual pathogens after treatment. At this regard, a TaqMan Real Time PCR method developed for the quantification of Leishmania infantum minicircle DNA in peripheral blood of naturally infected dogs sampled before and at different time points after the beginning of a standard antileishmanial therapy will be illustrated.     

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.     

Evaluation of new linkers and synthetic methods for internal modified oligonucleotides

Several fluorescence resonance energy transfer (FRET) oligonucleotide probes were made with different internal linkages between the DNA and the quencher dye. In one example, a 5'-fluorescein beta-actin-based 26-mer DNA sequence was synthesized bearing an internal Tamra quencher. Two different versions were prepared using either conventional C5 [N-(6-aminohexyl)-3-acrylamido]pyrimidine-modified uridine and solution-phase Tamra active ester coupling or solid-phase addition of a Tamra amidite to a C5 [N-(6-hydroxyhexyl)-3-acrylamido]pyrimidine-modified uridine. The products were compared in functional assays. They performed very similarly both in a fluorescence-based melting point assay as well as in quantitative PCR. Another set of beta-actin probes were synthesized utilizing N4 [N-2-(ethylene glycol ethyl)-5-methyl]cytidine and solid-phase Tamra amidite addition at positions flanking those of the uridine. These versions gave lower T(m)s than either uridine-labeled probe and did not work as well in quantitative PCR. A control experiment using oligonucleotides with the same modified residues but without fluorophores attached revealed the same trend as the T(m) study of internal Tamra-labeled probes. Experimental details for the synthesis, purification, and testing are presented.     

Profiling DNA methylation by melting analysis

The idea of modifying DNA with bisulfite has paved the way for a variety of polymerase chain reaction (PCR) methods for accurately mapping 5-methylcytosine at specific genes. Bisulfite selectively deaminates cytosine to uracil under conditions where 5-methylcytosine remains unreacted. Following conventional PCR amplification of bisulfite-treated DNA, original cytosines appear as thymine while 5-methylcytosines appear as cytosine. Because the relative thermostability of a DNA duplex increases with increasing content of G:C base pairs, PCR products originating from DNA templates with different contents of 5-methylcytosine differ in melting temperature, i.e., the temperature required to convert the double helix into random coils. We describe two methods that resolve differentially methylated DNA sequences on the basis of differences in melting temperature. The first method integrates PCR amplification of bisulfite-treated DNA and subsequent melting analysis by using a thermal cycler coupled with a fluorometer. By including in the reaction a PCR-compatible, fluorescent dye that specifically binds to double-stranded DNA, the melting properties of the PCR product can be examined directly in the PCR tube by continuous fluorescence monitoring during a temperature transition. The second method relies on resolution of alleles with different 5-methylcytosine contents by analysis of PCR products in a polyacrylamide gel containing a gradient of chemical denaturants. Optimal resolution of differences in melting temperature is achieved by a special design of PCR primers. Both methods allow resolution of "heterogeneous" methylation, i.e., the situation where the content and distribution of 5-methylcytosine in a target gene differ between different molecules in the same sample.     

Allelic discrimination by nick-translation PCR with fluorogenic probes.

Nick-translation PCR was performed with fluorogenic probes. Two probes were used: one complementary to a sequence containing the F508 codon of the normal human cystic fibrosis (CF) gene (wt DNA) and one complementary to a sequence containing the delta F508 three base pair deletion (mut DNA). Each probe contained a unique and spectrally resolvable fluorescent indicator dye at the 5' end and a common quencher dye attached to the seventh nucleotide from the 5' end. The F508/delta F508 site was located between the indicator and quencher. The probes were added at the start of a PCR containing mut DNA, wt DNA or heterozygous DNA and were degraded during thermal cycling. Although both probes were degraded, each probe generated fluorescence from its indicator dye only when the sequence between the indicator and quencher dyes was perfectly complementary to target. The identify of the target DNA could be determined from the post-PCR fluorescence emission spectrum.     

Neospora caninum: quantification of DNA in the blood of naturally infected aborted and pregnant cows using real-time PCR

This study quantified Neospora caninum DNA in the blood and brain of pregnant and aborted heifers by monitoring PCR product formation in real-time using SYBR Green I, a double-stranded DNA-binding dye. Primers were designed to amplify a 188 bp product specific to N. caninum from the Nc-5 gene fragment of N. caninum. Similarly, a 71 bp product was amplified from the 28S rRNA gene of bovine genomic DNA that served as a control. Agarose gel electrophoresis and analysis of the melting curve for PCR products showed that both primer pairs were specific to their targets. Standard curves were generated for both bovine and N. caninum genomic DNA, and were used to compute the relative concentration of parasite to bovine DNA in the test samples. The concentration of N. caninum DNA in 1 ng of bovine genomic DNA obtained from blood ranged between 0.097 ng at the 1st week of the observation and 0 ng at the 15th week in aborted cows. In pregnant cows, the values ranged between 0.080 ng at the 1st week and 0.155 ng at the 15th week of observation. There was a sustained decrease of DNA concentration in the aborted group after abortion and an increase in DNA concentration in the pregnant group. Comparison of parasite DNA in blood and brain of infected heifers showed a higher DNA concentration in brain than in blood. This study shows that N. caninum DNA can be quantified to obtain the relative concentration of parasite DNA to host genomic DNA in blood. This technique allows testing of a large number of samples at one time and can be done without the need for slaughter of tested animals.