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.
Amplicon melting is a closed-tube method for genotyping that does not require probes , real-time analysis , or allele-specific polymerase chain reaction. However , correct differentiation of homozygous mutant and wild-type samples by melting temperature (T m ) requires high-resolution melting and closely controlled reaction conditions. When three different DNA extraction methods were used to isolate DNA from whole blood , amplicon T m differences of 0.03 to 0.39°C attributable to the extractions were observed. To correct for solution chemistry differences between samples , complementary unlabeled oligonucleotides were included as internal temperature controls to shift and scale the temperature axis of derivative melting plots. This adjustment was applied to a duplex amplicon melting assay for the methylenetetrahydrofolate reductase variants 1298A>C and 677C>T. Highand low-temperature controls bracketing the amplicon melting region decreased the T m SD within homozygous genotypes by 47 to 82%. The amplicon melting assay was 100% concordant to an adjacent hybridization probe (HybProbe) melting assay when temperature controls were included , whereas a 3% error rate was observed without temperature correction. In conclusion , internal temperature controls increase the accuracy of genotyping by high-resolution amplicon melting and should also improve results on lower resolution instruments. Amplicon melting analysis is a simple closed-tube genotyping method that uses a saturating DNA binding dye instead of fluorescently labeled primers or probes.1 Highresolution melting analysis can detect single base changes and other variations in single or multiplex polymerase chain reaction (PCR).2 Wild-type and homozygous mutant samples typically have sharp, symmetric melting transitions, whereas heterozygous samples have more complex, gradual melting curves. Homozygous sequence changes result in characteristic shifts in melting temperature (T m ). [2][3][4][5] In contrast, heterozygous samples are identified by melting peak shape and width and not by T m . Correct identification of sample genotype by amplicon melting requires standardization of reaction conditions to achieve reproducible, characteristic melting profiles. Reaction conditions can vary between lots of PCR reagents, including different buffers introduced by the DNA isolation method. Ionic strength, in particular, significantly affects T m . -10The current study introduces the use of one or more internal controls for temperature calibration between reactions. Complimentary, unlabeled oligonucleotides that do not interfere with the PCR were designed so that they melt outside the temperature region of PCR product melting. Any buffer differences that affect duplex T m s affects both the amplicon and the internal temperature controls, allowing subsequent temperature correction of melting profiles. As a genotyping target, the 1298AϾC and 677CϾT variants of the methylenetetrahydrofolate reductase (MTHFR) gene were used. A single-color duplex amplicon melting assay (with a...
High-resolution melting techniques are a simple and cost-effective alternative to other closed-tube genotyping methods. Here, we genotyped human platelet antigens (HPAs) 1 to 6 and 15 by high-resolution melting methods that did not require labeled probes. Conventional melting analysis with hybridization probes (HybProbes) was also performed at each locus. HybProbe assays were performed individually, whereas amplicon melting (HPAs 1 to 5 and 16) and unlabeled probe (HPA 6) assays were duplexed when possible. At all loci for each method, both homozygous and heterozygous genotypes were easily identified. We analyzed 100 blinded clinical samples (33 amniotic fluid, 12 cultured amniocytes, and 55 blood samples) for all 7 single-nucleotide polymorphisms (SNPs) by each method. Genotype assignments could be made in 99.0% of the SNPs by high-resolution melting and in 98.7% of the SNPs with HybProbes with an overall genotype concordance of 98.8%. Errors included two sample misidentifications and six incorrect assignments that were all resolved by repeating the analysis. Advantages of high-resolution melting include rapid assay development and execution, no need for modified oligonucleotides, and similar accuracy in genotyping compared with other closed-tube melting methods. (J Mol
The measurement of multiple antigens in a single sample poses clinical and methodological challenges. Here we describe the validation of a multiplexed sandwich enzyme-linked immunosorbent assay (ELISA) array (microELISA) of nine antigens. The antigens tested simultaneously were: alpha-fetoprotein (AFP), prostate specific antigen (PSA), carcinoembryonic antigen (CEA), cancer antigen 125 (CA 125), CA 15-3, CA 19-9, beta-human chorionic gonadotropin (beta-hCG), luteinizing hormone (LH), and follicle stimulating hormone (FSH). At least 44 clinical samples were tested for each antigen. microELISA results for the nine antigens were then compared with clinical laboratory results obtained for the same antigens in individual chemiluminescent immunoassays. The microELISA had a coefficient of variation (cv) of 7.3% within an assay and 12.6% for assays run at different times. A statistical comparison of results from the microELISA with results from the clinical laboratory showed that the assays had correlation coefficients ranging from 0.99 to 0.76, and Deming regression demonstrated that four of the nine assays were high-quality assays and not statistically different to the individual assays. To determine if the differences in the assays were due to methodology, the microELISA was also compared with conventional ELISAs using identical antibodies and reagents. Deming regression demonstrated that five of the eight assays were high-quality, indicating that a poor correlation between a microELISA and an individual immunoassay are partly due to antibody differences.
Large B‐cell lymphoma with IRF4 rearrangement is a provisional entity in the 2017 World Health Organization classification. In order to characterize these lymphomas in children from the United States, IRF4 FISH and immunohistochemical stains were performed on 32 follicular lymphoma and diffuse large B‐cell lymphoma (DLBCL) from Children's Oncology Group studies. Two DLBCLs (6%) had IRF4 rearrangements, one involving the ileocecal valve and another involving the tonsil and cerebrospinal fluid. Both cases had strong, diffuse IRF4/MUM1 immunohistochemical staining, which may be a pathologic clue to the diagnosis. Reclassification of these cases may have prognostic and therapeutic implications.
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