No abstract

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...

Background: DNA hairpins have been used in molecular analysis of PCR products as self-probing amplicons. Either physical separation or fluorescent oligonucleotides with covalent modifications were previously necessary. Methods: We performed asymmetric PCR for 40–45 cycles in the presence of the saturating DNA dye, LCGreen Plus, with 1 primer including a 5′ tail complementary to its extension product, but without any special covalent modifications. Samples were amplified either on a carousel LightCycler for speed or on a 96/384 block cycler for throughput. In addition to full-length amplicon duplexes, single-stranded hairpins were formed by the primer tail “snapping back” and hybridizing to its extension product. High-resolution melting was performed on a HR-1 (for capillaries) or a LightScanner (for plates). Results: PCR products amplified with a snapback primer showed both hairpin melting at lower temperature and full-length amplicon melting at higher temperature. The hairpin melting temperature was linearly related to the stem length (6–28 bp) and inversely related to the log of the loop size (17–135 bases). We easily genotyped heterozygous and homozygous variants within the stem, and 100 blinded clinical samples previously typed for F5 1691G>A (Leiden) were completely concordant by snapback genotyping. We distinguished 7 genotypes in 2 regions of CFTR exon 10 with symmetric PCR using 2 snapback primers followed by product dilution to favor intramolecular hybridization. Conclusions: Snapback primer genotyping with saturating dyes provides the specificity of a probe with only 2 primers that are free of special covalent labels in a closed-tube system.

No abstract

No abstract