66
Y). One primer for the X allele is set to include X′ at the 3′ end (antisense), where
X′ is the antisense of X, with the counterpart sense primer upstream. For the Y
allele, a sense primer including Y at the 3′ end is set, with the antisense primer
downstream. One common band and one specifi c band for each allele are amplifi ed,
which allows genotyping directly by electrophoresis. This method is exemplifi ed by
application to the polymorphisms of beta-adrenoceptor 2 and interleukin 1B. It is
simpler than RFLP (restriction fragment length polymorphism), which requires
incubation with a restriction enzyme.
TaqMan Real-Time PCR
TaqMan (Life Technologies/Thermo Fisher) is a real-time PCR method that enables
one-step mutation detection. In PCR, forward and reverse primers hybridize to a
specifi c sequence of the target DNA in order to amplify the target sequence. The
TaqMan probe, with its bound fl uorophore and quencher, hybridizes to a second
target sequence within the amplifi ed product. When the PCR product is further
amplifi ed in subsequent cycles, the AmpliTaq enzyme cleaves the TaqMan probe (5′
nuclease activity) so that it can continue to copy its target sequence.
The reporter dye and quencher dye are separated, resulting in increased fl uores-
cence of the reporter. This process occurs in every amplifi cation cycle and does not
interfere with the exponential accumulation of product. Because release of the
reporter dye is associated with the amplifi cation of the specifi c gene DNA, the fl uo-
rescent signal is generated only if the gene sequence is present in the sample.
The TaqMan method can detect as few as fi ve copies of target in a background of
500 ng of DNA. The risk of PCR contamination is eliminated with this technology
because detection occurs within the amplifi cation reaction, thus also eliminating
postamplifi cation analysis costs.
Locked Nucleic Acid
Locked Nucleic Acids (LNA) is a novel oligonucleotide analogue containing a con-
formationally restricted nucleotide with a 2′-O, 4′-C-methylene bridge that induces
unprecedented thermal affi nities when mixed with complementary single stranded
DNA and RNA. LNA combines the highest affi nity ever reported for a DNA analog
for complementary DNA and RNA with a superb ability to discriminate between
correct and incorrect target sequences. This property is extremely important for
diagnostic use. An example of application of LNA technology for personalized
medicine is that it has been used successfully been to develop ELISA-assays that
detect the Apolipoprotein B R3500Q mutation. This facilitates a rational screening
of patients with cardiovascular disease for abnormalities in levels and metabolism
of lipoproteins.
2 Molecular Diagnostics in Personalized Medicine