DNA melting analysis.

Melting 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.

Rapid Cycle and Extreme Polymerase Chain Reaction.

Rapid cycle polymerase chain reaction (PCR) amplifies DNA in 10-30 min, while extreme PCR is complete in less than 1 min. These methods do not sacrifice quality for speed; sensitivity, specificity, and yield are equivalent or better than conventional PCR. What is required (and not widely available) is rapid, accurate control of reaction temperature during cycling. Specificity improves with cycling speed, and efficiency can be maintained by increasing polymerase and primer concentrations. Speed is aided by simplicity, dyes that stain double-stranded DNA are less expensive than probes, and one of the simplest polymerases, the deletion mutant KlenTaq, is used throughout. Rapid amplification can be coupled with endpoint melting analysis to verify product identity. Instead of commercial master mixes, detailed formulations for reagents and master mixes compatible with rapid cycle and extreme PCR are described.

Nearest-neighbour transition-state analysis for nucleic acid kinetics.

We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl2 (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = -0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl2 compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.

Retrospective clinical trial experimentally validates glioblastoma genome-wide pattern of DNA copy-number alterations predictor of survival.

Modeling of genomic profiles from the Cancer Genome Atlas (TCGA) by using recently developed mathematical frameworks has associated a genome-wide pattern of DNA copy-number alterations with a shorter, roughly one-year, median survival time in glioblastoma (GBM) patients. Here, to experimentally test this relationship, we whole-genome sequenced DNA from tumor samples of patients. We show that the patients represent the U.S. adult GBM population in terms of most normal and disease phenotypes. Intratumor heterogeneity affects ≈ 11 % and profiling technology and reference human genome specifics affect <1% of the classifications of the tumors by the pattern, where experimental batch effects normally reduce the reproducibility, i.e., precision, of classifications based upon between one to a few hundred genomic loci by >30%. With a 2.25-year Kaplan-Meier median survival difference, a 3.5 univariate Cox hazard ratio, and a 0.78 concordance index, i.e., accuracy, the pattern predicts survival better than and independent of age at diagnosis, which has been the best indicator since 1950. The prognostic classification by the pattern may, therefore, help to manage GBM pseudoprogression. The diagnostic classification may help drugs progress to regulatory approval. The therapeutic predictions, of previously unrecognized targets that are correlated with survival, may lead to new drugs. Other methods missed this relationship in the roughly 3B-nucleotide genomes of the small, order of magnitude of 100, patient cohorts, e.g., from TCGA. Previous attempts to associate GBM genotypes with patient phenotypes were unsuccessful. This is a proof of principle that the frameworks are uniquely suitable for discovering clinically actionable genotype-phenotype relationships.

Reverse transcriptase kinetics for one-step RT-PCR.

Understanding reverse transcriptase (RT) activity is critical for designing fast one-step RT-PCRs. We report a stopped-flow assay that monitors SYBR Green I fluorescence to investigate RT activity in PCR conditions. We studied the influence of PCR conditions on RT activity and assessed the accuracy of cDNA synthesis predictions for one-step RT-PCR. Nucleotide incorporation increased from 26 to 89 s-1 between 1.5 and 6 mM MgCl2 but was largely unaffected by changes in KCl. Conversely, increasing KCl from 15 to 75 mM increased apparent rate constants for RT-oligonucleotide binding (0.010-0.026 nM-1 s-1) and unbinding (0.2-1.5 s-1). All rate constants increased between 22 and 42 °C. When evaluated by PCR quantification cycle, cDNA predictions differed from experiments using RNase H+ RT (average 1.7 cycles) and RNase H- (average 4.5 cycles). Decreasing H+ RT concentrations 10 to 104-fold from manufacturer recommendations improved cDNA predictions (average 0.8 cycles) and increased RT-PCR assay efficiency. RT activity assays and models can be used to aid assay design and improve the speed of RT-PCRs. RT type and concentration must be selected to promote rapid cDNA synthesis but minimize nonspecific amplification. We demonstrate 2-min one-step RT-PCR of a Zika virus target using reduced RT concentrations and extreme PCR.

Removal of artifact bias from qPCR results using DNA melting curve analysis.

Quantitative PCR (qPCR) allows the precise measurement of DNA concentrations and is generally considered to be straightforward and trouble free. However, analyses using validated Sybr Green I-based assays regularly amplify both the correct product and an artifact. Amplification of more than 1 product can be recognized when melting curve analysis is performed after the qPCR. Currently, such reactions need to be excluded from further analysis because the quantification result is considered meaningless. However, when the fraction of the fluorescence associated with the correct product can be determined, the quantitative result of the qPCR analysis can be corrected. The main assumptions of this correction model are: 1) the melting peak of the correct product can be identified, 2) the PCR efficiencies of all amplified products are similar, 3) the relative size of the melting peaks reflects the relative concentrations of the products, and 4) the relative concentrations do not change as the reaction reaches plateau. These assumptions were validated in a series of model experiments. The results show that the quantitative results can be corrected. Implementation of a correction for the presence of artifact amplification in the analysis of qPCR data leads to more reliable quantitative results in qPCR experiments.-Ruijter, J. M., Ruiz-Villalba, A., van den Hoff, A. J. J., Gunst, Q. D., Wittwer, C. T., van den Hoff, M. J. B. Removal of artifact bias from qPCR results using DNA melting curve analysis.

The kinetic requirements of extreme qPCR.

The kinetic requirements of quantitative PCR were experimentally dissected into the stages of DNA denaturation, primer annealing, and polymerase extension. The temperature/time conditions for 2 stages were kept optimal, while the other was limited until the amplification efficiency decreased as measured by an increase in quantification cycle (Cq). Extension was studied in a commercial capillary LightCycler®. Using a rapid deletion mutant of Taq (KlenTaq™), about 1 s was required for every 70 bp of product length. To study annealing and denaturation times of <1 s, a custom "extreme" PCR instrument with 3 temperatures was used along with increased primer and polymerase concentrations. Actual sample temperatures and times were measured rather than programmed or predicted. For denaturation, 200-500 ms above the denaturation threshold was necessary for maximal efficiency. For annealing, 300-1000 ms below the annealing threshold was required. Temperature thresholds were set at 98% primer annealing or PCR product denaturation as determined experimentally by melting curves. Progressing from rapid cycle PCR to extreme PCR decreased cycling times by 10-60 fold. If temperatures are controlled accurately and flexibility in reagents is allowed, PCR of short products can be performed in less than 15 s. We also put PCR in context to other emerging methods and consider its relevance to the evolution of molecular diagnostics.

Evaluation of Mass Cytometry in the Clinical Laboratory.

BACKGROUND: Mass cytometry can differentiate more channels than conventional flow cytometry. However, for clinical use, standardization and agreement with well-established methods is paramount. We compared mass cytometry to standard clinical flow cytometry. METHODS: Mass and flow cytometry were performed in parallel on peripheral blood samples from 25 healthy individuals. Antibody staining was performed on the same samples at the same time, and analyzed for granulocyte, monocyte, lymphocyte, T, B, NK, CD4 and CD8 percentages. Validation parameters included comparison to flow cytometry, inter- and intra-assay precision and establishment of reference intervals. RESULTS: There was a positive correlation between mass and flow cytometry for the eight populations studied (R2 between 0.26 and 0.97). Slopes of the best-fit lines varied from 0.50 to 1.21 (fluorescence/mass). No significant differences in variance were found (F-test, P > 0.05). However, paired t-tests were significantly different for four of the eight markers (granulocytes, NK cells, T cells and CD4 cells), resulting in different reference intervals. Signal intensities were correlated for monocytes, lymphocytes, T, CD4 and CD8 cells (R2 = 0.41-0.57). The mass cytometry intra-assay precisions were 0.7-8.5% and inter-assay precisions 1.5-13.8%. CONCLUSION: Mass and flow cytometry evaluations of whole blood for major cell populations correlate with similar precision and signal intensity. However, for clinical use, separate reference interval studies are required. Cell population identification should rely on gating strategies that take advantage of the characteristics offered by each method. © 2019 International Clinical Cytometry Society.

Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds.

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.

A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood.

BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of ≥1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.

The Effect of Single Mismatches on Primer Extension.

BACKGROUND: Allele-specific PCR is an important diagnostic tool that identifies single-nucleotide variants by preferential amplification of a particular allele, using primers that are mismatched to all but one allele variant. METHODS: We applied a fluorescent stopped-flow polymerase assay to measure extension rates from oligonucleotide hairpins to simulate primer-template pairs. Under PCR-applicable conditions, reaction rates were recorded in nucleotides per second per polymerase (nt/s/poly). The effects of temperature, potassium chloride, mismatch type, and position were studied with primarily a deletion mutant of Thermus aquaticus (Taq) DNA polymerase and 135 oligonucleotide sequences. RESULTS: Rates at 65 °C were between 205 ± 11 and 177 ± 8 nt/s/poly for matched templates and between 4.55 ± 0.21 and 0.008 ± 0.005 nt/s/poly for 3'-mismatched templates. Although extension rates progressively increased with mismatches further away from the 3' end, rates were still reduced by as much as 84% with a C · C mismatch 6 bases from the 3' end. The optimal extension temperature for matched sequences was 70 °C, shifting to 55-60 °C for 3' mismatches. KCl inhibited mismatch extension. The Michaelis constant (Km) was increased and the apparent unimolecular rate constant (kcat) decreased for 3' mismatches relative to matched templates. CONCLUSIONS: Although primer extension of mismatches depends on mismatch type and position, variation also depends on local sequence, KCl concentration, and the type of polymerase. Introduction of 3' mismatches reduces the optimal temperature for extension, suggesting higher annealing temperatures for better allele discrimination. Quantitative descriptions of expected specificity in allele-specific PCR provide additional design direction and suggest when other methods (e.g., high-resolution melting analysis) may be a better choice.

High-Speed Melting Analysis: The Effect of Melting Rate on Small Amplicon Microfluidic Genotyping.

BACKGROUND: High-resolution DNA melting analysis of small amplicons is a simple and inexpensive technique for genotyping. Microfluidics allows precise and rapid control of temperature during melting. METHODS: Using a microfluidic platform for serial PCR and melting analysis, 4 targets containing single nucleotide variants were amplified and then melted at different rates over a 250-fold range from 0.13 to 32 °C/s. Genotypes (n = 1728) were determined manually by visual inspection after background removal, normalization, and conversion to negative derivative plots. Differences between genotypes were quantified by a genotype discrimination ratio on the basis of inter- and intragenotype differences using the absolute value of the maximum vertical difference between curves as a metric. RESULTS: Different homozygous curves were genotyped by melting temperature and heterozygous curves were identified by shape. Technical artifacts preventing analysis (0.3%), incorrect (0.06%), and indeterminate (0.4%) results were minimal, occurring mostly at slow melting rates (0.13-0.5 °C/s). Genotype discrimination was maximal at around 8 °C/s (2-8 °C/s for homozygotes and 8-16 °C/s for heterozygotes), and no genotyping errors were made at rates >0.5 °C/s. PCR was completed in 10-12.2 min, followed by melting curve acquisition in 4 min down to <1 s. CONCLUSIONS: Microfluidics enables genotyping by melting analysis at rates up to 32 °C/s, requiring <1 s to acquire an entire melting curve. High-speed melting reduces the time for melting analysis, decreases errors, and improves genotype discrimination of small amplicons. Combined with extreme PCR, high-speed melting promises nucleic acid amplification and genotyping in < 1 min.

Gain-of-function EGLN1 prolyl hydroxylase (PHD2 D4E:C127S) in combination with EPAS1 (HIF-2α) polymorphism lowers hemoglobin concentration in Tibetan highlanders.

Tibetans have lived at high altitude for generations and are thought to be genetically adapted to hypoxic environments. Most are protected from hypoxia-induced polycythemia, and a haplotype of EPAS1, encoding hypoxia-inducible factor (HIF-2α), has been associated with lower hemoglobin levels. We earlier reported a Tibetan-specific EGLN1 haplotype encoding PHD2 which abrogates HIF augmentation in hypoxia. We genotyped 347 Tibetan individuals from varying altitudes for both the Tibetan-specific EGLN1 haplotype and 10 candidate SNPs in the EPAS1 haplotype and correlated their association with hemoglobin levels. The effect of the EGLN1 haplotype on hemoglobin exhibited age dependency at low altitude, while at higher altitudes, it showed a trend to lower hemoglobin levels in the presence of the Tibetan-selected EPAS1 rs142764723 C/C allele. The observed gene-environment and gene-gene interactions and the moderate effect of the EGLN1 and EPAS1 haplotypes on hemoglobin indicate that other modifiers exist. It remains to be determined whether a blunting of erythropoiesis or other physiological consequences of HIF downregulation are the primary drivers of these genetic adaptations among Tibetans. KEY MESSAGE: Most Tibetans are protected from polycythemia while living in high altitude. An EGLN1 co-adapted haplotype, EGLN1 c.12C>G, c.380G>C is uniquely Tibetan. The Tibetan EPAS1 haplotype has introgressed from the Denisovan genome. While EGLN1 and EPAS1 genotypes lower Hb, this study indicates additional Hb modifiers.

Genomic Copy Number Analysis of HER2-Equivocal Breast Cancers.

OBJECTIVES: Guidelines for HER2 testing define an equivocal range for HER2 using two approved testing methods, immunohistochemistry (IHC) and in situ hybridization (ISH). We investigated genome-wide copy number alterations in this subgroup. METHODS: Ten breast cancers with equivocal HER2 status by both IHC and ISH were analyzed by single-nucleotide polymorphism cytogenomic microarray (SNP array). DNA ploidy analysis by flow cytometry was performed on nine cases with sufficient material remaining. RESULTS: SNP array analysis showed uniform gain of chromosome 17 (polysomy) in one case and segmental copy number gains encompassing HER2 and the centromere in five other cases. Flow cytometry revealed hyperdiploidy in six cases, all but one of which also had HER2 gains on SNP array. Although there was no evidence of HER2 amplification by SNP array, six cases showed amplification of other genomic regions, including known oncogenes in four cases. CONCLUSIONS: A combination of hyperdiploidy and segmental copy number gains contributes to HER2 ISH-equivocal results in most breast cancers. Cases in which HER2 copy number gain is not corroborated by genomic analysis suggest the presence of other contributing variables influencing ISH results. Genomic copy number analysis also implicates non-HER2 oncogenic drivers in many cases that are HER2 equivocal.

Copy number assessment by competitive PCR with limiting deoxynucleotide triphosphates and high-resolution melting.

BACKGROUND: DNA copy number variation is associated with genetic disorders and cancer. Available methods to discern variation in copy number are typically costly, slow, require specialized equipment, and/or lack precision. METHODS: Multiplex PCR with different primer pairs and limiting deoxynucleotide triphosphates (dNTPs) (3-12 µmol/L) were used for relative quantification and copy number assessment. Small PCR products (50-121 bp) were designed with 1 melting domain, well-separated Tms, minimal internal sequence variation, and no common homologs. PCR products were displayed as melting curves on derivative plots and normalized to the reference peak. Different copy numbers of each target clustered together and were grouped by unbiased hierarchical clustering. RESULTS: Duplex PCR of a reference gene and a target gene was used to detect copy number variation in chromosomes X, Y, 13, 18, 21, epidermal growth factor receptor (EGFR), survival of motor neuron 1, telomeric (SMN1), and survival of motor neuron 2, centromeric (SMN2). Triplex PCR was used for X and Y and CFTR exons 2 and 3. Blinded studies of 50 potential trisomic samples (13, 18, 21, or normal) and 50 samples with potential sex chromosome abnormalities were concordant to karyotyping, except for 2 samples that were originally mosaics that displayed a single karyotype after growth. Large cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7) (CFTR) deletions, EGFR amplifications, and SMN1 and SMN2 copy number assessments were also demonstrated. Under ideal conditions, copy number changes of 1.11-fold or lower could be discerned with CVs of about 1%. CONCLUSIONS: Relative quantification by restricting the dNTP concentration with melting curve display is a simple and precise way to assess targeted copy number variation.