Biomedical Engineering Reference
In-Depth Information
11.2. Restriction of PCR Products
Restriction mapping is a common way to verify the identity of a PCR prod-
uct. It is also a simple method of detecting restriction site polymorphisms and
of detecting mutations that are associated with the creation or destruction of
restriction sites. There is no need to purify the PCR product prior to restriction,
and most restriction enzymes are functional in a restriction mix in which the
PCR product constitutes up to one-half of the total volume.
11.3. Sequence-Specific Oligonucleotide Hybridization
Sequence-specific oligonucleotide hybridization is a powerful method for
detecting the presence of sequence polymorphisms in a region amplified by
PCR. Short oligonucleotides are synthesized and labeled (either radioactively
or nonradioactively), allowed to hybridize to dot blots of the PCR products
(
8
)
,
and washed under conditions that allow the discrimination of a single nucle-
otide mismatch between the probe and the target PCR product.
For the detection of a range of DNA polymorphisms at a given locus, the
hybridization can be performed “in reverse,” that is, with the oligonucleotides
immobilized onto the filter. Labeled amplified products from target DNA are
then hybridized to the filters and washed under appropriate conditions
(
18
)
.
When the oligonucleotides are immobilized onto a glass slide, it can be
miniaturized to form a microarray. Labeled amplified products from DNA can
be hybridized to the microarray for the investigation of chromosomal aberra-
tions
(
19-21
)
.
11.4. Cloning of PCR Product
PCR products may be cloned easily using conventional recombinant DNA
technology. To facilitate cloning of PCR products into vectors, restriction sites
may be incorporated into the primer sequences. Digestion of the PCR products
with the appropriate restriction enzymes will then allow “sticky end” ligation
into similarly restricted vector DNA.
11.5. Real-Time PCR
A fluorescent probe hybridizing to a region between the 3' ends of the prim-
ers can be incorporated into the PCR reaction
(
22
)
. In a particular incarnation of
this technique, during each round of PCR, the fluorescent probes would be
cleaved and the detectable fluorescent signal would increase. The amount of
increased fluorescent signal is proportional to the number of newly synthesized
amplicons. A major advantage of real-time PCR is that it can be used to deter-
mine the amount of initial templates.
