Biomedical Engineering Reference
In-Depth Information
given by the equation
x
(l + E)
n
,
where
x
= initial amount of target,
E
= effi-
ciency of amplification, and
n
= number of PCR cycles. After a few cycles, the
resulting product is of the size determined by the distance between the 5' ends
of the two primers. With the performance of a previous reverse transcription
step, PCR can also be applied to RNA.
3. Composition of the PCR
Conventional PCR is usually performed in a volume of 10-100
L.
Deoxynucleoside triphosphates (dATP, dCTP, dGTP, and dTTP) at a concen-
tration of 200
µ
M
each, 10 to 100 pmol of each primer, the appropriate salts,
buffers, and DNA polymerase are included. Many manufacturers have included
reaction buffer with their DNA polymerase, and this proves convenient for
newcomers to the PCR process. With the development of nanotechnology, there
is a trend for the miniaturization of the PCR reaction. PCR reaction has been
reported to have been successfully performed in an 86-pL microchamber
(
3
)
.
µ
4. Primers
Primers are designed to flank the sequence of interest. Oligonucleotide prim-
ers are usually between 18 and 30 bases long, with a GC content of about 50%.
Complementarity at the 3' ends of the primers should be avoided in order to
decrease the likelihood of forming the primer-dimer artifact. Runs of three or
more Cs or Gs at the 3' ends of the primers should be avoided in order to
decrease the probability of priming GC-rich sequences nonspecifically. A num-
ber of computer programs that assist primer design are available. However, for
most applications, PCR is sufficiently forgiving in that most primer pairs seem
to work. The primers are generally positioned between 100 and 1000 bp apart.
It should be noted, however, that for high-sensitivity applications, shorter PCR
products are preferred. For most applications, purification of the PCR primers
is not necessary. To simplify subsequent operations, it is recommended that all
primers be diluted to the same concentration (e.g., 50 pmol/
L) so that the
same volume of each primer is required for each reaction. Some primer pairs
seem to fail without any obvious reason, and when difficulty arises, one simple
solution is to change one or both of the primers.
Several sets of primers can be included in a single PCR reaction chamber for
the simultaneous amplification of different targets from a common DNA
source. In order to achieve similar efficiencies in all PCR reactions, the melt-
ing temperatures of different sets of primers should be as close as possible. The
melting temperature is defined as the temperature at which one-half of the
oligos form a duplex with the complementary sequence. There are a wide range
of applications for this multiplex PCR technique, e.g., detection of infectious
agents
(
4
)
and oncogene mutations
(
5
)
.
µ
