Background: High-resolution melting of PCR amplicons with the DNA dye LCGreen TM I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR. We adapted this system to genotype single-nucleotide polymorphisms (SNPs) after rapid-cycle PCR (12 min) of small amplicons (<50 bp). Methods: Engineered plasmids were used to study all possible SNP base changes. In addition, clinical protocols for factor V (Leiden) 1691G>A, prothrombin 20210G>A, methylenetetrahydrofolate reductase (MTHFR) 1298A>C, hemochromatosis (HFE) 187C>G, and -globin (hemoglobin S) 17A>T were developed. LCGreen I was included in the reaction mixture before PCR, and high-resolution melting was obtained within 2 min after amplification. Results: In all cases, heterozygotes were easily identified because heteroduplexes altered the shape of the melting curves. Approximately 84% of human SNPs involve a base exchange between A::T and G::C base pairs, and the homozygotes are easily genotyped by melting temperatures (T m s) that differ by 0.8 -1.4°C. However, in ϳ16% of SNPs, the bases only switch strands and preserve the base pair, producing very small T m differences between homozygotes (<0.4°C). Although most of these cases can be genotyped by T m , one-fourth (4% of total SNPs) show nearest-neighbor symmetry, and, as predicted, the homozygotes cannot be resolved from each other. In these cases, adding 15% of a known homozygous genotype to unknown samples allows melting curve separation of all three genotypes.
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.
High resolution melting is a new method of genotyping and variant scanning that can be seamlessly appended to PCR amplification. Limitations of genotyping by amplicon melting can be addressed by unlabeled probe or snapback primer analysis, all performed without labeled probes. High resolution melting can also be used to scan for rare sequence variants in large genes with multiple exons and is the focus of this article. With the simple addition of a heteroduplex-detecting dye before PCR, high resolution melting is performed without any additions, processing or separation steps. Heterozygous variants are identified by atypical melting curves of a different shape compared to wild type homozygotes. Homozygous or hemizygous variants are detected by prior mixing with wild type DNA. Design, optimization, and performance considerations for high resolution scanning assays are presented for rapid turnaround of gene scanning. Design concerns include primer selection and predicting melting profiles in silico. Optimization includes temperature gradient selection of the annealing temperature, random population screening for common variants, and batch preparation of primer plates with robotically deposited and dried primer pairs. Performance includes rapid DNA preparation, PCR, and scanning by high resolution melting that require, in total, only 3 hours when no variants are present. When variants are detected, they can be identified in an additional 3 hours by rapid cycle sequencing and capillary electrophoresis. For each step in the protocol, a general overview of principles is provided, followed by an in depth analysis of one example, scanning of CYBB, the gene that is mutated in X-linked chronic granulomatous disease.
Legionella spp. are a common cause of community-acquired respiratory tract infections and an occasional cause of nosocomial pneumonia. A PCR method for the detection of legionellae in respiratory samples was evaluated and was compared to culture. The procedure can be performed in 6 to 8 h with a commercially available DNA extraction kit (Qiagen, Valencia, Calif.) and by PCR with gel detection. PCR is performed with primers previously determined to amplify a 386-bp product within the 16S rRNA gene of Legionella pneumophila. We can specifically detect the clinically significant Legionella species including L. pneumophila, L. micdadei, L. longbeachae, L. bozemanii, L. feeleii, and L. dumoffii. The assay detects 10 fg (approximately two organisms) of legionella DNA in each PCR. Of 212 clinical specimens examined by culture, 100% of the culture-positive samples (31 of 31) were positive by this assay. By gel detection of amplification products, 12 of 181 culture-negative samples were positive forLegionella species by PCR, resulting in 93% specificity. Four of the 12 samples with discrepant results (culture negative, PCR positive) were confirmed to be positive for Legionellaspecies by sequencing of the amplicons. The legionella-specific PCR assay that is described demonstrates high sensitivity and high specificity for routine detection of legionellae in respiratory samples.
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