Regular ArticleProduct Differentiation by Analysis of DNA Melting Curves during the Polymerase Chain Reaction☆
Abstract
A microvolume fluorometer integrated with a thermal cycler was used to acquire DNA melting curves during polymerase chain reaction by fluorescence monitoring of the double-stranded DNA specific dye SYBR Green I. Plotting fluorescence as a function of temperature as the thermal cycler heats through the dissociation temperature of the product gives a DNA melting curve. The shape and position of this DNA melting curve are functions of the GC/AT ratio, length, and sequence and can be used to differentiate amplification products separated by less than 2°C in melting temperature. Desired products can be distinguished from undesired products, in many cases eliminating the need for gel electrophoresis. Analysis of melting curves can extend the dynamic range of initial template quantification when amplification is monitored with double-stranded DNA specific dyes. Complete amplification and analysis of products can be performed in less than 15 min.
References (26)
- T. Ishiguro et al.
Anal. Biochem.
(1995) - M. Hiyoshi et al.
Anal. Biochem.
(1994) - J. Yguerabide et al.
Anal. Biochem.
(1995) - Y. Maeda et al.
J. Mol. Biol.
(1990) - H.A. Erlich et al.
Science
(1991) - C.T. Wittwer et al.
BioTechniques
(1991) - R. Higuchi et al.
Bio/Technology
(1993) - C.T. Wittwer et al.
BioTechniques
(1997) - C.T. Wittwer et al.
BioTechniques
(1997) - C.T. Wittwer et al.
Gene Quantification
(1997)
Cited by (1313)
DNA melting analysis
2024, Molecular Aspects of MedicineMelting is a fundamental property of DNA that can be monitored by absorbance or fluorescence. PCR conveniently produces enough DNA to be directly monitored on real-time instruments with fluorescently labeled probes or dyes. Dyes monitor the entire PCR product, while probes focus on a specific locus within the amplicon. Advances in amplicon melting include high resolution instruments, saturating DNA dyes that better reveal multiple products, prediction programs for domain melting, barcode taxonomic identification, high speed microfluidic melting, and highly parallel digital melting. Most single base variants and small insertions or deletions can be genotyped by high resolution amplicon melting. High resolution melting also enables heterozygote scanning for any variant within a PCR product. A web application (uMelt, http://www.dna-utah.org) predicts amplicon melting curves with multiple domains, a useful tool for verifying intended products. Additional applications include methylation assessment, copy number determination and verification of sequence identity. When amplicon melting does not provide sufficient detail, unlabeled probes or snapback primers can be used instead of covalently labeled probes. DNA melting is a simple, inexpensive, and powerful tool with many research applications that is beginning to make its mark in clinical diagnostics.
mtDNA-triggered pH response signal-amplified fluorescent probe for multiple cell discrimination
2024, Chinese Chemical LettersDeveloping fluorescence probes with multiple responses has vital significance but remains challenging. Herein, for the first time, we present a mitochondrial DNA (mtDNA)-triggered pH response signal-amplified fluorescent probe (QCY-DBT) for multiple cell detection. The probe exhibited a large stokes shift (229 nm), excellent DNA selectivity over RNA, and ultrasensitivity of detection limit (DL; 74.0 ng/mL). Thus, QCY-DBT was successfully applied to analyze multiple human peripheral blood cells and visualize mtDNA in healthy and apoptotic cells. In the tumor acidic environment (pH 6.0–7.0), the absorbance of QCY-DBT at 436 nm increased, and the fluorescence signal (665 nm) was amplified by mtDNA, which enabled the direct observation of tumor cells. Our study provides help in designing smart probes with multiple responses for efficient abnormal cell detection.
Effective attenuation of extracellular antibiotic resistance gene risks in wastewater by capacitive deionization
2024, Journal of Environmental Chemical EngineeringDespite the effective inactivation on antibiotic resistant bacteria, current water and wastewater treatment technologies are often ineffective in removing antibiotic resistance genes (ARGs), which remain active outside cells, known as extracellular ARGs (eARGs). eARGs can persist for a long time and facilitate bacterial transformation in receiving environments, thereby, posing a threat to public health. This study applied capacitive deionization (CDI), as a promising desalination technology, for eARGs removal from wastewater. Results showed that CDI using carbon electrodes was significantly more effective than activated carbon adsorption in removing eARGs, with the removal efficiency and rate constant that are 2.1–11.2 times (p < 0.05) and 2.5–13 times (p < 0.05) higher. In the flow-by mode, CDI achieved the highest removal of eARGs (3.5 log) in just 15 min. Furthermore, CDI treatment significantly decreased the frequency of horizontal gene transfer (HGT) and increased potential for natural degradation, thereby reducing the risk of eARGs propagation. The high electric field (>1000 V/m) applied in CDI induces DNA conformational damage and DNA sequence mutations, which are the main mechanisms for removing eARG. This study will provide technical guidance and theoretical support for the removal of eARG in wastewater.
Improving the efficiency of Y-chromosome detection and the quality of STR typing in forensic casework with an in-house made qPCR and HRM system based on SYTO™ 9 chemistry
2024, Forensic Science InternationalDNA quantification prior to STR amplification is a crucial step in forensic casework. Obtaining good-quality genetic STR profiles depends mainly on the amount and integrity of the DNA input in the PCR. In addition, the detection of male trace DNA provides key information for forensic investigation.
To evaluate the correlation between the quantification results obtained with the previously developed Amel-Y system, and its ability to detect Y-chromosome DNA by HRM, with the resulting STR profiles, and to ultimately show that Amel-Y can be routinely used in forensic casework to improve STR and Y-STR results.
Biological samples derived from forensic casework (85 reference and 391 evidence samples) were quantified by the Amel-Y system (a duplex qPCR/HRM based on SYTO™ 9 chemistry) using Rotor-Gene 6000. STRs were amplified and analyzed with GeneAmp™ PCR System 9700 or Veriti™ Thermal Cyclers and ABI 3500 Genetic Analyzer, respectively.
After DNA normalization, a total of 386 STR profiles were obtained (305 full and 81 partial). Sex typing by HRM was 100% successful in reference samples. Male DNA was detected by HRM in 210 evidence samples. 80/201 were mixed with an excess of female DNA. In addition, Amel-Y was able to detect Y-chromosome DNA in mixed samples that did not amplify the Y-variant of Amelogenin marker with commercial STR kits. The reproducibility and precision of the Amel-Y system were demonstrated (CVCt% ≤ 9.55) within the dynamic range analyzed (0.016–50 ng/µL; 41 independent runs). Amel-Y also proved to be compatible with other real-time PCR platforms.
We demonstrated that Amel-Y is a robust quantification system that can be routinely used in forensic casework to obtain reliable autosomal STR profiles and can be suitable as a predictor for Y-STR typing success when male DNA is detected. HRM can be used as a rapid screening tool for male DNA detection in mixed samples. Alternative designs like Amel-Y offer independence from commercial quantification kits in forensic labs.
Fabrication and characterization of bee pollen extract nanoparticles: Their potential in combination therapy against human A549 lung cancer cells
2023, Food Hydrocolloids for HealthBee pollen extract (BPE) based polymer nanoparticles (BPENP) were fabricated in the current study by using bovine serum albumin (BSA) and the complex was then coated by folic acid conjugated protamine to be targeted for cancer cells. Spectroscopic and microscopic methods were used to characterize the resultant nanoparticles. High flavonoid and polyphenolic contents were detected by HPLC. At 336 nm, it was found that BPENP provided the highest absorption by UV. The average particle size ranged between 25 and 40 nm. The inhibitory effects and IC50 values of the acquired BPE, BPENP, Avastin, or their combination at 24 and 48 h were detected against A549 lung cancer cells. The combination therapy assay of BPENP and Avastin revealed a very significant synergism between the two drugs with a low combination index (CI). These therapies particularly the combination therapy have significantly affected the expression of the HRAS, MAPK, and apoptotic genes such as Bax, Bcl-2, and Caspase 3. It was established that obtained BPENP might be employed to support the administration of Avastin for the treatment of lung cancer.
New genetic markers for 100% arabica coffee demonstrate high discriminatory potential for InDel-HRM-based coffee authentication
2023, Food Research InternationalFood authenticity is crucial in today's society, given the heightened consumer awareness and attention to the products they consume. Reliable and efficient techniques are needed to quickly detect potential food adulterations that can negatively impact product quality and economic value. Coffee, a globally traded agricultural product, holds immense economic importance, with an estimated value of USD 83 billion. It is widely consumed and recognized as a functional food that provides minerals (K, Mg, Mn, Cr), niacin, and antioxidants. However, the preferred coffee species, Coffea arabica, known for its superior drink quality, is often adulterated with Coffea canephora (Robusta and Conilon) beans, even in 100% Arabica coffee. To distinguish between these two coffee species, a comprehensive study was conducted using a robust approach to identify differences in Single-Ortholog Copy (SOC) based on InDel regions in these gene pairs. These differences were validated using a meticulous methodology that considered variations in amplicon size: electrophoretic profile, and high-resolution melting (HRM). The innovative combination of InDels and HRM resulted in highly distinctive HRM profiles, outperforming SNP-based methods previously used. The targeted InDel approach utilized in this study facilitated precise quantification of Coffea species beans with a detection sensitivity of 0.5%. The study's findings establish the reliability and accuracy in distinguishing between the two coffee species, showcasing the valuable application of InDels for quality control and ensuring the authenticity of coffee beans. This pioneering research contributes to the advancement of authenticity verification methods for both imported and exported coffee beans, as well as in future studies that require significant genetic differences between these species, such as C. arabica and C. canephora.
- ☆
F. Ferre, Ed.