RESUMO
BACKGROUND: Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS: A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS: Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 µmol/L) and polymerase (0.45 U/µL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS: Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.
Assuntos
DNA Bacteriano/análise , DNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Clostridioides difficile/genética , Variações do Número de Cópias de DNA , DNA Bacteriano/metabolismo , DNA Viral/metabolismo , Genótipo , Vírus da Hepatite B/genética , Humanos , Microfluídica , Transição de Fase , Fatores de Tempo , Temperatura de TransiçãoRESUMO
The ability to accurately monitor solution temperature is important for the polymerase chain reaction (PCR). Robust amplification during PCR is contingent on the solution reaching denaturation and annealing temperatures. By correlating temperature to the fluorescence of a passive dye, noninvasive monitoring of solution temperatures is possible. The temperature sensitivity of 22 fluorescent dyes was assessed. Emission spectra were monitored and the change in fluorescence between 45 and 95°C was quantified. Seven dyes decreased in intensity as the temperature increased, and 15 were variable depending on the excitation wavelength. Sulforhodamine B (monosodium salt) exhibited a fold change in fluorescence of 2.85. Faster PCR minimizes cycling times and improves turnaround time, throughput, and specificity. If temperature measurements are accurate, no holding period is required even at rapid speeds. A custom instrument using fluorescence-based temperature monitoring with dynamic feedback control for temperature cycling amplified a fragment surrounding rs917118 from genomic DNA in 3min and 45s using 35 cycles, allowing subsequent genotyping by high-resolution melting analysis. Gold-standard thermocouple readings and fluorescence-based temperature differences were 0.29±0.17 and 0.96±0.26°C at annealing and denaturation, respectively. This new method for temperature cycling may allow faster speeds for PCR than currently considered possible.
Assuntos
Corantes Fluorescentes/química , Reação em Cadeia da Polimerase , DNA/metabolismo , Genética Forense , Genoma Humano , Humanos , Polimorfismo de Nucleotídeo Único , Rodaminas/química , Temperatura de TransiçãoRESUMO
Fluorescent high-resolution DNA melting analysis is a robust method of genotyping and mutation scanning. However, removing background fluorescence is important for accurate classification and to correctly display helicity. Linear baseline extrapolation, commonly used with absorbance, often fails at low temperatures when fluorescence is used. A new quantum method of background removal based on the inherent decrease of fluorescence with temperature is described. Absorbance and fluorescence melting curves were compared using synthetic targets including hairpins, unlabeled probes, and a 50 bp duplex. In addition, the quantum method was compared to a previously described exponential method for analysis of genotyping data produced after polymerase chain reaction (PCR), including those from small amplicons, unlabeled probes, and snapback primers. The quantum method best matched absorbance data and predicted helicity, with the exponential method displaying low-temperature bulges and domain artifacts that can lead to incorrect genotyping. When two melting domains were widely separated, quantum analysis produced a flat baseline between domains, while exponential analysis was temperature-dependent. Both methods have little effect on the melting temperature (Tm) although some differences were significant (hairpin Tm values increased 0.7 °C by the quantum method and decreased 1.5 °C by exponential method, p = 0.01). However, peak heights on derivative plots were strongly algorithm-dependent, with exponential analysis enhancing low-temperature peaks while dampening high-temperature peaks. Quantum-analyzed fluorescence curves were a better match to absorbance data in terms of shape, area, and peak height compared to other methods, indicating that DNA helicity is best approximated by the quantum method.
Assuntos
DNA/química , DNA/genética , Técnicas de Genotipagem/métodos , Artefatos , Sequência de Bases , Desnaturação de Ácido Nucleico , Reação em Cadeia da Polimerase , Espectrometria de Fluorescência , Temperatura de TransiçãoRESUMO
Accurate control of the sample temperature during thermal cycling is critical for successful polymerase chain reaction (PCR). Direct sensor contact with the reaction is problematic, forcing measurements external to the sample and compromising accuracy during rapid temperature transitions. The widespread use of fluorescence in real-time PCR and melting analysis suggests another measure of temperature, the intrinsic fluorescence of temperature-sensitive passive dyes. Calibration curves correlating sulforhodamine B fluorescence to temperature on nine real-time PCR instruments were obtained by heating at 0.018-0.1 °C/s between 50 and 95 °C, with a twofold change in fluorescence. After instrument stabilization for 20 min, no dye photobleaching was observed and thermal degradation was 2.2%/h at 80 °C. During cycling, solution temperatures derived from fluorescence were well matched to thermocouples placed within samples, but not to temperatures recorded by the instrument. Solution temperatures lagged instrument temperatures by up to 8 °C during cycling, often requiring 5-10 s at target temperatures for equilibration. Melting curves were displaced by 0.2-1.1 °C. Temperature inaccuracies were dependent on the instrument, the ramp rate, and the sample volume. The fluorescence of passive dyes can be used to accurately assess solution temperatures during PCR and should be particularly useful at fast cycling speeds.
Assuntos
DNA/metabolismo , Corantes Fluorescentes/química , Reação em Cadeia da Polimerase em Tempo Real , Rodaminas/química , Regiões 3' não Traduzidas , Transição de Fase , Temperatura de TransiçãoRESUMO
Among nucleic acid analytical methods, high-resolution melting analysis is gaining more and more attention. High-resolution melting provides simple, homogeneous solutions for variant scanning and genotyping, addressing the needs of today's overburdened laboratories with rapid turnaround times and minimal cost. The flexibility of the technique has allowed it to be adopted by a wide range of disciplines for a variety of applications. In this review we examine the broad use of high-resolution melting analysis, including gene scanning, genotyping (including small amplicon, unlabeled probe and snapback primers), sequence matching and methylation analysis. Four major application arenas are examined to demonstrate the methods and approaches commonly used in particular fields. The appropriate usage of high-resolution melting analysis is discussed in the context of known constraints, such as sample quality and quantity, with a particular focus placed on proper experimental design in order to produce successful results.