Biomedical Engineering Reference
In-Depth Information
Fig. 5. Four modes of single nucleotide polymorphism genotyping by melting analy-
sis. The traditional hybridization probe design (top row) uses a pair of probes, one
labeled with an acceptor fluorophore (encircled A) and the other with a donor
fluorophore (encircled D). The single-hybridization-probe design (second row) lacks
the second probe. The amplicon melting design (third row) uses a saturating double-
stranded DNA binding dye. The two homozygotes differ in melting temperature and
the heterozygote has an additional low-temperature transition caused by heteroduplexes.
The unlabeled probe design (bottom row), similar to amplicon melting, does not require
a covalently attached fluorescent label and uses a DNA binding dye. However, because
a probe is used, the derivative melting curves are better separated than with amplicon
melting. Homozygous G allele (dashed line in far right column), homozygous A allele
(dotted line), and the GA heterozygote (solid line). (Adapted from
ref.
17
with the
permission of Elsevier Press.)
0.3°C/s and high-resolution melting techniques may be necessary. When probes
are used, the melting rate is usually 0.1°C and high resolution is not needed.
Melting analysis using unlabeled probes and the dye LCGreen I is especially
attractive because no covalently labeled oligonucleotides are required
(
7
)
.
