Biology Reference
In-Depth Information
Electrochemical biosensors of single nucleotide polymorphisms
(SNP,point mutations) can use
(i) different stabilities of duplexes displaying full complemen-
tarity between the probe and tDNA (homoduplexes between
wild-type probe and wild-type tDNA or mutant probe and
mutant target) and those involving mismatched nucleotides
(heteroduplexes between wild-type probe and mutant target,
or vice versa) [13]. Discrimination of perfectly matched and
mismatched duplexes can be achieved by performing DNA
hybridization at stringent conditions achieved by elevated
temperature and decreased ionic strength or via applying
a peptide nucleic acid probe instead of DNA. Under opti-
mum conditions, the homoduplex gives positive hybridization
response, while the heteroduplex is not stable, thus giving a
signal-off response to the mutation in one of the hybridizing
strands.
(ii) primer extension incorporation of a labeled nucleotide within
theSNPsite[30].Thetargettemplateisannealedwithaprimer
complementary to the target segment “upstream” (relative to
DNApolymerasecatalyzedelongationoftheprimerthatalways
proceedsinthe5'
→
3'direction)tothepositionofinterest,and
a labeled dNTP (e.g., with biotin to attach an enzyme in the
followingstep,orwitharedoxmarker)isaddedtothereaction
mixture. Under proper conditions, the labeled nucleotide is
attached to the primer only when it is complementary to
the base at the first “free” position. Using different labels for
differentnucleotides,allfourpossiblebaseswithintheSNPsite
can beprobed in asingle reaction.
(iii) electronic properties of the duplex DNA and perturbations
in the DNA electronic properties in the presence of single
base mismatches [34]. Disruption of
π
-stacks within the DNA
double helix due to presence of the mismatch has been shown
to prevent DNA-mediated charge transfer between electrode
and an intercalator bound at the opposite (relative to the
electrode surface) end of the double helix, which was e
cient
in the perfectly matched (and perfectly base-pair-stacked)
homoduplex.
