Systems & Accessories Overview
The WAVE System allows you to discover and detect mutations
and size nucleic acid fragments through denaturing high performance liquid
chromatography
(DHPLC).
The WAVE System is more accurate and sensitive
than other methods for mutation detection.
The DHPLC Process
Wild and test or mutant type alleles are amplified separately,
mixed, heated and then cooled to form homoduplexes and heteroduplexes (if
a mutation is present). The diagram below depicts this process.
When a mutation is present, there are four possible configurations:
The sample
containing homoduplexes and heteroduplexes (if a mutation is present) is
injected into the buffer flow path containing triethyammonium acetate
(TEAA) and acetonitrile (ACN). In solution, the TEAA forms the positively
charged triethylammonium ion (TEA+) that has both hydrophobic and hydrophilic
ends. The DNASep cartridge is located in the oven and contains beads that
are hydrophobic. The positively charged portion of theTEA+ forms an association
with the negatively charged
phosphate
backbone of the DNA creating a hydrophobic "fur" on the fragment. This entire hydrophobic entity then behaves as would a typical hydrophobic molecule and is attracted to the hydrophobic beads.
The fragment
specific methods created by
Navigator™ Software control both the temperature
of the
oven and the ACN gradient. The concentration of ACN increases over time
based on this method. As the ACN concentration increases bridging capabilities
of the TEA+ ions decrease and the DNA fragments are released from
the cartridge. Heteroduplexes, with mismatched base pairs, elute off of the
cartridge first followed by the homoduplexes. The fragments pass through
the UV detector which detects the absorbance over time. This information
is sent to Navigator Software on the computer. It records the results in
the form of a chromatogram. If no mutation is present, all of the homoduplex
DNA fragments elute off of the cartridge at the same time producing a single
peak on the chromatogram. If a mutation is present then two to four peaks
will be visible as illustrated below.
At non-denaturing temperatures, the smaller fragments elute off of the cartridge before larger fragments allowing for fragment separation based on size.
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