Luming Zhou

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.

Detection of c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution amplicon melting analysis

High-resolution amplicon melting analysis was used to scan for c-kit-activating mutations in exons 9, 11, 13, and 17 in 29 neoplasms diagnosed as gastrointestinal stromal tumors (GISTs). Immunohistochemically, 7 of 29 did not show strong CD 17 positivity and might represent true smooth muscle tumors or c-kit-negative GISTs. No c-kit-activating mutations were detected in the 7 CD117- cases by high-resolution amplicon melting analysis or direct DNA sequencing. Alterations in the remaining 22 CD117+ cases included 13 (59%) in exon 11, 2 (9%) in exon 9, 1 (5%) in exon 13, and none in exon 17. The genetic alterations consisted of point mutations and in-frame insertions, duplications, and deletions. In exon 11, 7 (54%) of 13 alterations have not been described previously. In 2 cases, the identical exon 11 mutation was observed in the primary tumor and a metastatic/recurrent lesion. In all cases, direct DNA sequencing confirmed that polymerase chain reaction products with an abnormal melting curve contained a mutation and products with a normal melting curve, a normal DNA sequence. High-resolution melting analysis can be used to scan DNA for potential c-kit-activating mutations and can aid in the diagnosis of GISTs.

Rare allele enrichment and detection by allele-specific PCR, competitive probe blocking and melting analysis

Differential amplification of variant and wild-type alleles by PCR is often used for rare allele enrichment. We have combined allele-specific PCR, competitive probe blocking, asymmetric PCR, and me...

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.

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.

Allele-Specific PCR with Competitive Probe Blocking for Sensitive and Specific Detection of BRAF V600E in Thyroid Fine-Needle Aspiration Specimens

Objective: To detect BRAF V600E mutation in thyroid fine-needle aspiration (FNA) slides and needle rinses (NR). Study Design: Tumor-enriched DNA was extracted from FNA smears, formalin-fixed paraffin-embedded (FFPE) sections, or NR specimens from 37 patients with confirmed papillary thyroid carcinoma or benign findings. An allele-specific primer selectively amplified the 1799 T>A BRAF mutation while simultaneously blocking amplification of wild-type (WT) BRAF with an unlabeled probe during PCR. Mutation detection was accomplished by melting analysis of the probe. Results: Allele-specific/blocking probe PCR confirmed the BRAF mutation status for 20 of 24 paired FNA/FFPE samples previously tested by fluorescent probe real-time PCR. For the other 4 cases, the sensitive PCR method detected the BRAF mutation in all paired FNA/FFPE samples. Previously, the mutation had been detected in only the FFPE samples. The BRAF mutation was also detected in some NR specimens. Conclusion: Treatment of patients with thyroid nodules is guided by FNA biopsy, which can be scantly cellular, necessitating a sensitive test that can detect low levels of BRAF V600E mutation in a WT background. We report increased detection of BRAF V600E in FNA specimens using allele-specific/blocking probe PCR, which has an analytical sensitivity of 0.01%.

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.

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

BackgroundCopy 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.MethodsCompetitive 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.ResultsOne 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.ConclusionsLimited 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.

Abstract 4923: Detecting copy number variation using dNTP limited PCR and high-resolution melting

Background: Copy number variation (CNV) is a common type of genetic variation. About 13% of genes in the human genome have variation in copy number. Copy number alterations (CNAs), somatic changes to chromosome structure that result in gain or loss in copies of sections of DNA, are very common in cancer and associated with particular cancer types. In some cases such as EGFR and HER2, the copy number alterations have led to the identification of cancer-causing genes and suggested specific therapeutic approaches. Materials and Methods: Target and reference regions of genomic DNA were amplified together using a variety of amplification limiting PCR reagent and cycling protocols. Ratios of reference and copy number variants were maintained by limiting quantities of dNTPs and allowing PCR to plateau. Suitable fragments having single melting domains, well-separated Tms, and no common homologs were designed using uMelt melting curve prediction software. After PCR, high-resolution melting (fluorescence vs. temperature) data was acquired from double stranded DNA dye LCGreen Plus present in the reaction. MeltWizard 6 software was used to remove background, equalize negative derivative melting peaks corresponding to the reference segment, and identify and quantify samples with different CNV ratios from the amplitude of their target melting peaks. The protocols were optimized on samples with known ratios. Results: All proposed methods of limiting amplification (cycle number, dNTPs, Taq polymerase) were found to be capable of preserving initial copy ratios sufficiently to identify all common human copy number variation ratios when used in conjunction with high-resolution melting peak reference normalization. CNV ratios far beyond this range, as small as 2 : 2.125 (5.88%), were detectable using the most robust, simplest, and highest resolution method of limiting dNTPs at an optimal 3.25 uM. Duplex PCR of reference CFTR and target EGFR genes were used to detect the copy number variation. Copy number of EGFR in 8 NSCNC lung cancer and 41 colon cancer samples were detected with 6.25 uM dNTP. In 7 out of 8 of lung cancer and 7 out of 41 colon cancer samples, the copy number of EGFR is higher than normal copy 2. The Tm of the reference peak may be lower (duplication) or higher (deletion) than that of the CNV target without affecting the results. Conclusion: Copy number variation can be quantified using reaction limited multiplex PCR followed by high-resolution melting analysis. This simple closed-tube method is fast, economical, more accurate and less susceptible to contamination than other methods. Assays are easy to design, limiting dNTPs is simple, and the results are stable and reliable. After PCR, only 5 to 10 minutes of melting and analysis are required to obtain results. Citation Format: Luming Zhou, Bobert Palais, Carl Wittwer. Detecting copy number variation using dNTP limited PCR and high-resolution melting. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4923. doi:10.1158/1538-7445.AM2015-4923

Abstract A23: Rare allele enrichment by DNA melting analysis

Since the sequencing of the human genome and the development of high throughput genotyping techniques, many genes have been identified that are associated with a variety of diseases through their SNPs and mutations. For example, in lung cancer, mutations in the gene encoding epidermal growth factor reporter (EGFR) can confer either positive therapeutic response or drug resistance to tyrosine kinase inhibitors. Additionally, mutations in the BRAF gene are strongly associated with cancerous thyroid nodules. In the early and post-treatment stages of cancer or in prenatal diagnostics, the proportion of the mutant allele is typically low within a background of wild-type. Even after amplification by standard PCR, the low levels of the minority allele that are present in tumor tissue or in plasma circulating DNA are not sufficient to permit genotyping or sequencing. Mutation detection plays a key role in several areas of medicine including diagnosis, treatment and prognosis. In cancer-related gene mutation diagnostics, low-level ( A snapback primer is a tail at the 5′ end of a primer that is complementary to its own extension product. When intramolecular hybridization is favored over intermolecular pairing, a hairpin is formed whose stem melting behavior depends on sequence. Rapid cycle PCR with a short extension time (0 sec) and an extension temperature lower than the Tm of the hairpin of the wild-type, perfectly matched allele obstructs primer extension while mismatched alleles denature, thus providing the competitive advantage and preferential amplification of minority alleles. Minority alleles down to 0.1% (1:1000) can be detected. Even greater detection sensitivity can be achieved using allele specific PCR, probe blocking and melting analysis. Unlabeled probes, dual hybridization probes, or molecular beacons can be used. Allele-specific competitive blocker PCR (ACB-PCR) was modified by using asymmetric PCR and melting analysis to increase the sensitivity. The probe serves as both a blocker and produces the probe melting curve to implement detection. Mutation detection by probe melting curve analysis is more reliable than PCR quantification because only the specifically amplified allele produces the melting curve associated with the target. Quantitative PCR with dsDNA dyes cannot distinguish the intended PCR product from primer dimers and other non-specific PCR products. In contrast, when using probe melting curves to detect mutations, the melting temperature and overall melting profile of probe melting curves specifically identify the known mutant target. Additionally, based upon probe melting temperatures of wild-type and mutant hybrids, a precise annealing temperature between these Tms guarantees that the PCR selectively amplifies the minority allele. Low allele specific primer concentrations (asymmetric PCR) further increase the sensitivity and increase the probe signal allowing a sensitivity of 0.001% (1:105) mutant to wild-type genomic DNA, or just a single copy in a 500 ng genomic DNA. Methods that do not require labeled probes (snapback primer and unlabeled probes) can be used to selectively amplify minority alleles by methods that are rapid (less than 20 minutes), easy to design, inexpensive and reliable. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A23.