Genotyping – getting rid of labeled probesClosed-tube (homogeneous) genotyping methods have strong advantages in the clinical laboratory and for point-of-care diagnostics. Because no processing is required between amplification and analysis steps, the need for automation and risk of contamination are eliminated. A classical example of a closed-tube genotyping method is the Taqman probe system. Taqman probes are allele-specific, meaning that in order to genotype, two probes (one for the wild type and another for the mutant allele) are required. Unknown variants will not be detected, and the fluorescent signal resulting from amplification-dependent probe degradation is measured only once each cycle during annealing or extension phases of PCR. Despite these limitations, Taqman is used commonly in laboratory medicine.
Melting analysis, which is performed immediately after PCR, is inherently a more powerful way of genotyping than Taqman probes. Fluroescence signal is monitored over a range of temperatures, rather than at only a single temperature. Before the advent of high-resolution melting, genotyping by melting used labeled probes, but unlike Taqman, it was able to distinguish many different alleles in one operation (including unexpected sequence variants). Different alleles resulted in different probe melting temperatures (Tm), and heterozygous PCR products were easily distinguished from homozygous samples by a double peak on derivative melting curve plots.
Genotyping without probes (amplicon melting):High-resoltuion melting analysis enables homogeneous genotyping without probes, even when the sequence change is only a single base (see Fig 1). With saturation dyes, the PCR product (amplicon) is labeled along its entire length, so that all melting domains are detected. For single-base genotyping, heterozygotes are easy to identify because of the change in curve shape. Different heterozygotes within the same amplicon can often be distinguished from each other by differences in curve shape. Over 93% of unique heterozygotes are distinguishable by high-resolution melting.
Fig 1. Genotyping by amplicon melting. (A) Single-base genotyping by small amplicon melting. (B) Single-base genotyping of a large DNA fragment (544 bp) that melts in two domains.
Most but not all homozygotes can be distinguished by melting. About 4% of human single-base variants are predicted to have the same Tm by nearest-neighbor symmetry. In such a case, mixing of samples is necessary for complete genotyping. After PCR, a known homozygote is mixed into each unknown homozygote and the mixture melted again. Alternatively, a known genotype can be added into all samples before PCR and quantitative heteroduplex analysis performed.
Fig 2. Genotyping a tetranucleotide repeat.
(A) Normalized, temperature-shifted melting curves of locus D5S818 with the number of repeats indicated. (B) Derivative plots of the same samples. (C) Duplexes formed by the four genotypes tested. (click image to enlarge)
Deletions, insertsions and repeats sequences can also be detected by high-resolution melting (see Fig 2 for examples of tetranucleotide repeat variants).
Unlabeled probe genotyping:An interesting variation on genotyping with saturation dyes is to include an unlabeled probe. In addition to the full length PCR product, the probe produces additional melting data focused on the sequence under the probe. Unequal primer concentrations are used to generate one strand of DNA in excess. Some of the excess strand hybridizes to the complementary unlabeled probe. Both probe and amplicon duplexes will be bound by the dye, generating melting data for both the probe and the amplicon. Although not absolutely necessary, high-resolution melting improves the quality of the melting curves and allows more variants to be distinguished from each other. Probes can be designed to mask certain variants or segments by incorporating deletions, mismatches, or universal bases. Multiple unlabeled probes can interrogate different regions of the amplicon. Unlabeled probes are helpful when amplicon melting alone does not provide adequate detail in highly polymorphic regions, or when internal redundancy is required of the assay.
Fig 3. Simultaneous genotyping and scanning. (A) Composite normalized melting curve showing transitions in both the probe and the amplicon regions (factor V Leiden locus). (B) Derivative plot of the probe region from part A after background removal by exponential subtraction. See Zhou et al, 2005 for more details. (Click images to enlarge.)
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