At 32[degrees]C/s, heteroduplex and homoduplex
peaks merge into a single, broad peak because of low data density.
Amplified heterozygotes have a complex melting curve shape consisting of 2 homoduplexes
(wild type and mutant) and 2 heteroduplexes that are influenced by the class of single-nucleotide polymorphism and other variants within the amplicon.
Cases with >4 detectable species cannot be explained easily, because only 4 species are possible (2 homoduplexes
and 2 heteroduplexes).
As mentioned above, it is possible that high-abundance mutants may become less differentiated from the wild type if enrichment is quite strong and if their homoduplex
melting curves are comparable to the wild-type melting curve (4).
The altered melting curve shape is a result of the presence of both heteroduplex and homoduplex
amplicons in the PCR product, vs only homoduplexes
from normal samples.
Different homozygotes each produce only a single homoduplex
For dHPLC, it has been shown (1) that temperature conditions can be optimized by use of predictive software to improve the separation of heteroduplex and homoduplex
peaks and achieve high-resolution results.
It relies on the use of a hydrophobic column based on reversed-phase liquid chromatography for the separation of heteroduplex and homoduplex
DNA at specific optimized temperatures and has recently been used by several research groups to study mitochondrial DNA (mtDNA) mutation (1-6).
DHPLC separates heteroduplex and homoduplex
molecules by ion-pair chromatography under partially denaturing conditions.
The continuing development of instruments with finer temperature control and fluorescence acquisition will lead to increased detail derived from heteroduplex and homoduplex
contributions to the overall melting curve, providing even greater ability to identify unique sequence variants.
The resulting melting curve is a composite of both heteroduplex and homoduplex
The more stable homoduplex
polymorphism sample (GC base pair) was distinguished by a higher [T.sub.m] than the less stable wild-type sample (AT base pair) (11,12).