Carl T. Wittwer

The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments

BACKGROUND Currently, a lack of consensus exists on how best to perform and interpret quantitative real-time PCR (qPCR) experiments. The problem is exacerbated by a lack of sufficient experimental detail in many publications, which impedes a readers ability to evaluate critically the quality of the results presented or to repeat the experiments. CONTENT The Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines target the reliability of results to help ensure the integrity of the scientific literature, promote consistency between laboratories, and increase experimental transparency. MIQE is a set of guidelines that describe the minimum information necessary for evaluating qPCR experiments. Included is a checklist to accompany the initial submission of a manuscript to the publisher. By providing all relevant experimental conditions and assay characteristics, reviewers can assess the validity of the protocols used. Full disclosure of all reagents, sequences, and analysis methods is necessary to enable other investigators to reproduce results. MIQE details should be published either in abbreviated form or as an online supplement. SUMMARY Following these guidelines will encourage better experimental practice, allowing more reliable and unequivocal interpretation of qPCR results.

Continuous Fluorescence Monitoring of Rapid Cycle DNA Amplification

Rapid cycle DNA amplification was continuously monitored by three different fluorescence techniques. Fluorescence was monitored by (i) the double-strand-specific dye SYBR Green I, (ii) a decrease i...

High-Resolution Genotyping by Amplicon Melting Analysis Using LCGreen

BACKGROUND High-resolution amplicon melting analysis was recently introduced as a closed-tube method for genotyping and mutation scanning (Gundry et al. Clin Chem 2003;49:396-406). The technique required a fluorescently labeled primer and was limited to the detection of mutations residing in the melting domain of the labeled primer. Our aim was to develop a closed-tube system for genotyping and mutation scanning that did not require labeled oligonucleotides. METHODS We studied polymorphisms in the hydroxytryptamine receptor 2A (HTR2A) gene (T102C), beta-globin (hemoglobins S and C) gene, and cystic fibrosis (F508del, F508C, I507del) gene. PCR was performed in the presence of the double-stranded DNA dye LCGreen, and high-resolution amplicon melting curves were obtained. After fluorescence normalization, temperature adjustment, and/or difference analysis, sequence alterations were distinguished by curve shape and/or position. Heterozygous DNA was identified by the low-temperature melting of heteroduplexes not observed with other dyes commonly used in real-time PCR. RESULTS The six common beta-globin genotypes (AA, AS, AC, SS, CC, and SC) were all distinguished in a 110-bp amplicon. The HTR2A single-nucleotide polymorphism was genotyped in a 544-bp fragment that split into two melting domains. Because melting curve acquisition required only 1-2 min, amplification and analysis were achieved in 10-20 min with rapid cycling conditions. CONCLUSIONS High-resolution melting analysis of PCR products amplified in the presence of LCGreen can identify both heterozygous and homozygous sequence variants. The technique requires only the usual unlabeled primers and a generic double-stranded DNA dye added before PCR for amplicon genotyping, and is a promising method for mutation screening.

Rapid Cycle Real-Time PCR: Methods and Applications

for Genetics and Oncology Volume Rapid Cycle Real-Time PCR Methods and Applications.- I Methods Useful in Genetics and Oncology.- Quantification of Cytokine mRNAs in Human Myocardial Biopsy Samples by Real-Time Quantitative PCR Technology Using the LightCycler Instrument.- Quantitative Two-Step RT-PCR for the Detection of Human ABCA1 Transporter on LightCycler Using Hybridization Probes and External Standards.- Quantification of Human Genomic DNA Using Retinoic X Receptor B Gene.- Genotyping by Guanosine-Dependent Quenching of Single-Labeled Fluorescein Probes.- Limitations of Melting Curve Analysis Using SYBR Green I - Fragment Differentiation and Mutation Detection in the CFTR-Gene.- SYBR Green I Analysis of the Trinucleotide Repeat Responsible for Huntingtons Disease.- II Applications in Genetics.- Parallel Genotyping of Different Genes: A Rapid Real-Time PCR Approach.- Detection of a Single Base Substitution in Single Cells by Melting Peak Analysis Using Dual-Color Hybridization Probes.- Rapid Screening for Five Major Cystic Fibrosis Mutations by Melting Peak Analysis Using Fluorogenic Hybridization Probes.- LightCycler PCR for the Polymorphisms -308 and -238 in the TNF AIpha Gene and for the TNFB1/B2 Polymorphism in the LT Alpha Gene.- Rapid Genotyping of 2-bp and 9-bp Deletion Mutations Using the LightCycler.- Genotyping of the Methionine-Valine Polymorphism at Codon 129 of the Human Prion Protein by Melting Point Analysis of Fluorescently Labeled Hybridization Probes.- Rapid Detection of Missense Mutations in the Prostatic Steroid 5?-Reductase Gene Using Real-Time Fluorescence PCR and Melting Curve Analysis.- III Applications in Oncolon.- Analysis of Microsatellite Instability by Melting Peak Analysis with BAT26 and BAT25 Specific Fluorescence Hybridization Probes.- Two Color Multiplexing and Typing of Human Papillomavirus Types 16,18 and 45 on LightCycler.- Quantitative Analysis of AML1-ETO Fusion Transcripts in t(8 21) Positive AML Using Real-Time RT-PCR.- Rapid Quantitative Detection of Free Cancer Cells in the Peritoneal Cavity of Gastric Cancer Patients with Real-Time CEA RT-PCR Using Hybridization Probes.- Quantitative Measurement of the mRNA Expression of the Tumor-Associated Antigen PRAME by Real-Time RT-PCR Using LightCycler and SYBR Green I Technology.- Expression Analysis of Telomerase-Genes hTERT and hTR by Quantitative PCR on LightCycler.- Measurement of MDR1 Gene Expression by Real-Time Quantitative RT-PCR Using the LightCycler Instrument.

High-resolution DNA melting analysis for simple and efficient molecular diagnostics

High-resolution melting of DNA is a simple solution for genotyping, mutation scanning and sequence matching. The melting profile of a PCR product depends on its GC content, length, sequence and heterozygosity and is best monitored with saturating dyes that fluoresce in the presence of double-stranded DNA. Genotyping of most variants is possible by the melting temperature of the PCR products, while all variants can be genotyped with unlabeled probes. Mutation scanning and sequence matching depend on sequence differences that result in heteroduplexes that change the shape of the melting curve. High-resolution DNA melting has several advantages over other genotyping and scanning methods, including an inexpensive closed tube format that is homogenous, accurate and rapid. Owing to its simplicity and speed, the method is a good fit for personalized medicine as a rapid, inexpensive method to predict therapeutic response.

The Digital MIQE Guidelines: Minimum Information for Publication of Quantitative Digital PCR Experiments

There is growing interest in digital PCR (dPCR) because technological progress makes it a practical and increasingly affordable technology. dPCR allows the precise quantification of nucleic acids, facilitating the measurement of small percentage differences and quantification of rare variants. dPCR may also be more reproducible and less susceptible to inhibition than quantitative real-time PCR (qPCR). Consequently, dPCR has the potential to have a substantial impact on research as well as diagnostic applications. However, as with qPCR, the ability to perform robust meaningful experiments requires careful design and adequate controls. To assist independent evaluation of experimental data, comprehensive disclosure of all relevant experimental details is required. To facilitate this process we present the Minimum Information for Publication of Quantitative Digital PCR Experiments guidelines. This report addresses known requirements for dPCR that have already been identified during this early stage of its development and commercial implementation. Adoption of these guidelines by the scientific community will help to standardize experimental protocols, maximize efficient utilization of resources, and enhance the impact of this promising new technology.

HIGH-RESOLUTION DNA MELTING ANALYSIS: ADVANCEMENTS AND LIMITATIONS

Recent advances in fluorescent dyes, methods, instruments and software for DNA melting analysis have created versatile new tools for variant scanning and genotyping. High resolution melting analysis (HRM or HRMA) is faster, simpler, and less expensive than alternative approaches requiring separations or labeled probes. With the addition of a saturating dye before PCR followed by rapid melting analysis of the PCR products, the sensitivity of heterozygote scanning approaches 100%. Specificity can be increased by identifying common polymorphisms with small amplicon melting, unlabeled probes or snapback primers to decrease the sequencing burden. However, some homozygotes require mixing for identification. Furthermore, different heterozygotes may produce melting curves so similar to each other that, although they clearly vary from homozygous variants, they are not differentiated from each other. Nevertheless, the experimental return for minimal effort is great. This focus issue of Human Mutation includes a concise, timely review on high resolution melting, a comparison to denaturing gradient gel electrophoresis, integration with qPCR for copy number assessment, combined amplicon scanning and unlabeled probe genotyping from a single melting curve, and applications to the mitochondrial genome and to BRCA1. Hum Mutat 30, 857–859, 2009.

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.

High resolution melting applications for clinical laboratory medicine

Separation of the two strands of DNA with heat (melting) is a fundamental property of DNA that is conveniently monitored with fluorescence. Conventional melting is performed after PCR on any real-time instrument to monitor product purity (dsDNA dyes) and sequence (hybridization probes). Recent advances include high resolution instruments and saturating DNA dyes that distinguish many different species. For example, mutation scanning (identifying heterozygotes) by melting is closed-tube and has similar or superior sensitivity and specificity compared to methods that require physical separation. With high resolution melting, SNPs can be genotyped without probes and more complex regions can be typed with unlabeled hybridization probes. Highly polymorphic HLA loci can be melted to establish sequence identity for transplantation matching. Simultaneous genotyping with one or more unlabeled probes and mutation scanning of the entire amplicon can be performed at the same time in the same tube, vastly decreasing or eliminating the need for re-sequencing in genetic analysis. High resolution PCR product melting is homogeneous, closed-tube, rapid (1-5 min), non-destructive and does not require covalently-labeled fluorescent probes. In the clinical laboratory, it is an ideal format for in-house testing, with minimal cost and time requirements for new assay development.

Minimizing the time required for DNA amplification by efficient heat transfer to small samples

Hot-air temperature cycling of 1- to 10-microliters samples in glass capillary tubes can amplify DNA by the polymerase chain reaction in 15 min or less. A rapid temperature cycler of low thermal mass was constructed to change sample temperatures among denaturation, annealing, and elongation segments in a few seconds. After 30 cycles of 30 s each, a 536-bp beta-globin fragment of human genomic DNA was easily visualized with ethidium bromide on agarose gels. With rapid cycling, amplification yield depended on polymerase concentration. The time required for DNA amplification can be markedly reduced from prevailing protocols if appropriate equipment and sample containers are used for rapid heat transfer to the sample.