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.

Accurate diagnosis of spinal muscular atrophy and 22q11.2 deletion syndrome using limited deoxynucleotide triphosphates and high-resolution melting.

BACKGROUND: Copy number variation (CNV) has been implicated in the genetics of multiple human diseases. Spinal muscular atrophy (SMA) and 22q11.2 deletion syndrome (22q11.2DS) are two of the most common diseases which are caused by DNA copy number variations. Genetic diagnostics for these conditions would be enhanced by more accurate and efficient methods to detect the relevant CNVs. METHODS: Competitive PCR with limited deoxynucleotide triphosphates (dNTPs) and high-resolution melting (HRM) analysis was used to detect 22q11.2DS, SMA and SMA carrier status. For SMA, we focused on the copy number of SMN1 gene. For 22q11.2DS, we analyzed CNV for 3 genes (CLTCL1, KLHL22, and PI4KA) which are located between different region-specific low copy repeats. CFTR was used as internal reference gene for all targets. Short PCR products with separated Tms were designed by uMelt software. RESULTS: One hundred three clinical patient samples were pretested for possible SMN1 CNV, including carrier status, using multiplex ligation-dependent probe amplification (MLPA) commercial kit as gold standard. Ninety-nine samples consisting of 56 wild-type and 43 22q11.2DS samples were analyzed for CLTCL1, KLHL22, and PI4KA CNV also using MLPA. These samples were blinded and re-analyzed for the same CNVs using the limited dNTPs PCR with HRM analysis and the results were completely consistent with MLPA. CONCLUSIONS: Limited dNTPs PCR with HRM analysis is an accurate method for detecting SMN1 and 22q11.2 CNVs. This method can be used quickly, reliably, and economically in large population screening for these diseases.

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.

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.

Microfluidic genotyping by rapid serial PCR and high-speed melting analysis.

BACKGROUND: Clinical molecular testing typically batches samples to minimize costs or uses multiplex lab-on-a-chip disposables to analyze a few targets. In genetics, multiple variants need to be analyzed, and different work flows that rapidly analyze multiple loci in a few targets are attractive. METHODS: We used a microfluidic platform tailored to rapid serial PCR and high-speed melting (HSM) to genotype 4 single nucleotide variants. A contiguous stream of master mix with sample DNA was pulsed with each primer pair for serial PCR and melting. Two study sites each analyzed 100 samples for F2 (c.*97G>A), F5 (c.1601G>A), and MTHFR (c.665C>T and c.1286A>C) after blinding for genotype and genotype proportions. Internal temperature controls improved melting curve precision. The platform's liquid-handling system automated PCR and HSM. RESULTS: PCR and HSM were completed in a total of 12.5 min. Melting was performed at 0.5 °C/s. As expected, homozygous variants were separated by melting temperature, and heterozygotes were identified by curve shape. All samples were correctly genotyped by the instrument. Follow-up testing was required on 1.38% of the assays for a definitive genotype. CONCLUSIONS: We demonstrate genotyping accuracy on a novel microfluidic platform with rapid serial PCR and HSM. The platform targets short turnaround times for multiple genetic variants in up to 8 samples. It is also designed to allow automatic and immediate reflexive or repeat testing depending on results from the streaming DNA. Rapid serial PCR provides a flexible genetic work flow and is nicely matched to HSM analysis.

Genotyping accuracy of high-resolution DNA melting instruments.

BACKGROUND: High-resolution DNA melting is a closed-tube method for genotyping and variant scanning that depends on the thermal stability of PCR-generated products. Instruments vary in thermal precision, sample format, melting rates, acquisition, and software. Instrument genotyping accuracy has not been assessed. METHODS: Each genotype of the single nucleotide variant (SNV) (c.3405-29A>T) of CPS1 (carbamoyl-phosphate synthase 1, mitochondrial) was amplified by PCR in the presence of LCGreen Plus with 4 PCR product lengths. After blinding and genotype randomization, samples were melted in 10 instrument configurations under conditions recommended by the manufacturer. For each configuration and PCR product length, we analyzed 32-96 samples (depending on batch size) with both commercial and custom software. We assessed the accuracy of heterozygote detection and homozygote differentiation of a difficult, nearest-neighbor symmetric, class 4 variant with predicted ΔT(m) of 0.00 °C. RESULTS: Overall, the heterozygote accuracy was 99.7% (n = 2141), whereas homozygote accuracy was 70.3% (n = 4441). Instruments with single sample detection as opposed to full-plate imaging better distinguished homozygotes (78.1% and 61.8%, respectively, χ(2) P < 0.0005). Custom software improved accuracy over commercial software (P < 0.002), although melting protocols recommended by manufacturers were better than a constant ramp rate of 0.1 °C with an oil overlay. PCR products of 51, 100, 272, and 547 bp had accuracies of 72.3%, 83.1%, 59.8%, and 65.9%, respectively (P < 0.0005). CONCLUSIONS: High-resolution melting detects heterozygotes with excellent accuracy, but homozygote accuracy is dependent on detection mode, analysis software, and PCR product size, as well as melting temperature differences between, and variation within, homozygotes.

Symmetric snapback primers for scanning and genotyping of the cystic fibrosis transmembrane conductance regulator gene.

BACKGROUND: High-resolution melting of PCR products is an efficient and analytically sensitive method to scan for sequence variation, but detected variants must still be identified. Snapback primer genotyping uses a 5' primer tail complementary to its own extension product to genotype the resulting hairpin via melting. If the 2 methods were combined to analyze the same PCR product, the residual sequencing burden could be reduced or even eliminated. METHODS: The 27 exons and neighboring splice sites of the CFTR [cystic fibrosis transmembrane conductance regulator (ATP-binding cassette sub-family C, member 7)] gene were amplified by the PCR in 39 fragments. Primers included snapback tails for genotyping 7 common variants and the 23 CFTR mutations recommended for screening by the American College of Medical Genetics. After symmetric PCR, the amplicons were analyzed by high-resolution melting to scan for variants. Then, a 5-fold excess of H2O was added to each reaction to produce intramolecular hairpins for snapback genotyping by melting. Each melting step required <10 min. Of the 133 DNA samples analyzed, 51 were from CFTR patient samples or cell lines. RESULTS: As expected, the analytical sensitivity of heterozygote detection in blinded studies was 100%. Snapback genotyping reduced the need for sequencing from 7.9% to 0.5% of PCR products; only 1 amplicon every 5 patients required sequencing to identify nonanticipated rare variants. We identified 2 previously unreported variants: c.3945A>G and c.4243-5C>T. CONCLUSIONS: CFTR analysis by sequential scanning and genotyping with snapback primers is a good match for targeted clinical genetics, for which high analytical accuracy and rapid turnaround times are important.

Snapback primer genotyping of the Gilbert syndrome UGT1A1 (TA)(n) promoter polymorphism by high-resolution melting.

BACKGROUND: Gilbert syndrome, a chronic nonhemolytic unconjugated hyperbilirubinemia, is associated with thymine-adenine (TA) insertions in the UGT1A1 (UDP glucuronosyltransferase 1 family, polypeptide A1) promoter. The UGT1A1 promoter genotype also correlates with toxicity induced by the chemotherapeutic drug irinotecan. Current closed-tube assays for genotyping the UGT1A1 (TA)(n) promoter polymorphism require multiple labeled probes and/or have difficulty classifying the (TA)(5) and (TA)(8) alleles. METHODS: An unlabeled 5' extension on one primer that creates a hairpin after asymmetric PCR was used to develop a snapback primer high-resolution melting assay for the (TA)(n) polymorphism. A new method that plots the local deviation from exponential decay to improve genotype clustering was used to remove background fluorescence and to analyze the data. The snapback assay was compared with small-amplicon melting and fragment length analyses in a blinded study of DNA samples from 100 African Americans. RESULTS: Genotyping results obtained by small-amplicon melting and snapback primer melting were 83% and 99% concordant, respectively, with results obtained by fragment analysis. Reanalysis of the single discordant sample in the results of the snapback genotyping assay and the fragment analysis revealed an error in the fragment analysis. High-resolution melting was required for accurate snapback genotyping of the UGT1A1 (TA)(n) polymorphism. The 100% accuracy obtained with a capillary-based instrument fell to ≤81% with plate-based instruments. CONCLUSIONS: In contrast to small-amplicon genotyping, snapback primer genotyping can distinguish all UGT1A1 promoter genotypes. Rapid-cycle PCR combined with snapback primer analysis with only 2 unlabeled PCR primers (one with a 5' extension) and a saturating DNA dye can genotype loci with several alleles in <30 min.

Enrichment and detection of rare alleles by means of snapback primers and rapid-cycle PCR.

BACKGROUND: Selective amplification of minority alleles is often necessary to detect cancer mutations in clinical samples. METHODS: Minor-allele enrichment and detection were performed with snapback primers in the presence of a saturating DNA dye within a closed tube. A 5' tail of nucleotides on 1 PCR primer hybridizes to the variable locus of its extension product to produce a hairpin that selectively enriches mismatched alleles. Genotyping performed after rapid-cycle PCR by melting of the secondary structure identifies different variants by the hairpin melting temperature (T(m)). Needle aspirates of thyroid tissue (n = 47) and paraffin-embedded biopsy samples (n = 44) were analyzed for BRAF (v-raf murine sarcoma viral oncogene homolog B1) variant p.V600E, and the results were compared with those for dual hybridization probe analysis. Needle aspirates of lung tumors (n = 8) were analyzed for EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] exon 19 in-frame deletions. RESULTS: Use of 18-s cycles and momentary extension times of "0 s" with rapid-cycle PCR increased the selective amplification of mismatched alleles. A low Mg(2+) concentration and a higher hairpin T(m) relative to the extension temperature also improved the detection limit of mismatched alleles. The detection limit was 0.1% for BRAF p.V600E and 0.02% for EGFR exon 19 in-frame deletions. Snapback and dual hybridization probe methods for allele quantification of the thyroid samples correlated well (R(2) = 0.93) with 2 more BRAF mutations (45 and 43, respectively, of 91 samples) detected after snapback enrichment. Different EGFR in-frame deletions in the lung samples produced different hairpin T(m)s. CONCLUSIONS: Use of snapback primers for enrichment and detection of minority alleles is simple, is inexpensive to perform, and can be completed in a closed tube in <25 min.

Identifying common genetic variants by high-resolution melting.

BACKGROUND: Heteroduplex scanning techniques usually detect all heterozygotes, including common variants not of clinical interest. METHODS: We conducted high-resolution melting analysis on the 24 exons of the ACVRL1 and ENG genes implicated in hereditary hemorrhagic telangiectasia (HHT). DNA in samples from 13 controls and 19 patients was PCR amplified in the presence of LCGreen I, and all 768 exons melted in an HR-1 instrument. We used 10 wild-type controls to identify common variants, and the remaining samples were blinded, amplified, and analyzed by melting curve normalization and overlay. Unlabeled probes characterized the sequence of common variants. RESULTS: Eleven common variants were associated with 8 of the 24 HHT exons, and 96% of normal samples contained at least 1 variant. As a result, the positive predictive value (PPV) of a heterozygous exon was low (31%), even in a population of predominantly HHT patients. However, all common variants produced unique amplicon melting curves that, when considered and eliminated, resulted in a PPV of 100%. In our blinded study, 3 of 19 heterozygous disease-causing variants were missed; however, 2 were clerical errors, and the remaining false negative would have been identified by difference analysis. CONCLUSIONS: High-resolution melting analysis is a highly accurate heteroduplex scanning technique. With many exons, however, use of single-sample instruments may lead to clerical errors, and routine use of difference analysis is recommended. Common variants can be identified by their melting curve profiles and genotyped with unlabeled probes, greatly reducing the false-positive results common with scanning techniques.

Quantitative heteroduplex analysis for single nucleotide polymorphism genotyping.

High-resolution melting of polymerase chain reaction (PCR) products can detect heterozygous mutations and most homozygous mutations without electrophoretic or chromatographic separations. However, some homozygous single nucleotide polymorphism (SNPs) have melting curves identical to that of the wild-type, as predicted by nearest neighbor thermodynamic models. In these cases, if DNA of a known reference genotype is added to each unknown before PCR, quantitative heteroduplex analysis can differentiate heterozygous, homozygous, and wild-type genotypes if the fraction of reference DNA is chosen carefully. Theoretical calculations suggest that melting curve separation is proportional to heteroduplex content difference and that the addition of reference homozygous DNA at one seventh of total DNA results in the best discrimination between the three genotypes of biallelic SNPs. This theory was verified experimentally by quantitative analysis of both high-resolution melting and temperature-gradient capillary electrophoresis data. Reference genotype proportions other than one seventh of total DNA were suboptimal and failed to distinguish some genotypes. Optimal mixing before PCR followed by high-resolution melting analysis permits genotyping of all SNPs with a single closed-tube analysis.

Involvement of multiple signaling pathways in follicular lymphoma transformation: p38-mitogen-activated protein kinase as a target for therapy.

Follicular lymphoma (FL) is the most common form of low-grade non-Hodgkin's lymphoma. Transformation to diffuse large B cell lymphoma (DLBCL) is an important cause of mortality. Using cDNA microarray analysis we identified 113 transformation-associated genes whose expression differed consistently between serial clonally related samples of FL and DLBCL occurring within the same individual. Quantitative RT-PCR validated the microarray results and assigned blinded independent group of 20 FLs, 20 DLBCLs, and five transformed lymphoma-derived cell lines with 100%, 70%, and 100% accuracy, respectively. Notably, growth factor cytokine receptors and p38beta-mitogen-activated protein kinase (MAPK) were differentially expressed in the DLBCLs. Immunohistochemistry of another blinded set of samples demonstrated expression of phosphorylated p38MAPK in 6/6 DLBCLs and 1/5 FLs, but not in benign germinal centers. SB203580 an inhibitor of p38MAPK specifically induced caspase-3-mediated apoptosis in t(14;18)+/p38MAPK+-transformed FL-derived cell lines. Lymphoma growth was also inhibited in SB203580-treated NOD-SCID mice. Our results implicate p38MAPK dysregulation in FL transformation and suggest that molecular targeting of specific elements within this pathway should be explored for transformed FL therapy.