The Polymerase Chain Reaction
(PCR) in detail
A piece of dsDNA includes the target region of
interest [middle, base-paired DNA]. We draw it such that the Sense
Strand is on the bottom, with the 5'3' direction drawn left to right.
The reverse complement Partner Strand is drawn on the
top, with the 3'5'
direction drawn right to left.
The first denaturation step
separates the two strands. The first Annealing step allows the
Forward (5'F3') and and Reverse (3'R5') Primers to
anneal to their complementary sequences in the top
and bottom strands, respectively. The primers are on opposite
strands at either end of the intervening target region. The 5'3' orientation of the Primers is always anti-parallel
to the target strand 3'5' as
drawn. The first Extension step extends both primers in the 5'3' direction. Both produce 'long
products' as they extend indefinitely on the their
respective template strands. These 'long products' are
shown immediately above and below the dsDNA.
The second denaturation again
separates the two strands of the target DNA, with the
same result as in the first cycle. The 'long products'
from the first cycle have incorporated into their 5'
ends the sequences of the Forward or
Reverse Primers (shown
below and above the long products, respectively). The second
annealing allows the Forward
(5'-F-3') and Reverse
(3'-R-5') primers to anneal to their
complementary sequences on the long products. These second
extensions then proceeds just until they reach the 5'
ends of their respective long products. For the first time,
the rxn produces ssDNA products exactly as long as
the distance between the 5' ends of the two primers.
These 'unit length products' are shown
immediately above and below the two 'long products'.
The third denaturation separates
all previously paired strands. The third annealing allows
the Forward and
Reverse primers
to anneal to the 3' complementary ends of the two ssDNA
'unit length products'. For the first time, extension of
these primers produces dsDNA products of unit
length, with 3' ends complementary to the Forward primer
on one strand, and the Reverse primer
on the other. The PCR process has made the regions
under the primers at either end of the dsDNA product
identical to the primer sequences: naturally
occurring SNPs cannot be detected in those regions.
On the fourth and subsequent
cycles, the dsDNA PCR products are
denatured, Forward and
Reverse primers
anneal to the 3' ends of either strand, and new
strands are synthesized off each template to the limits of
the primer. The number of PCR products doubles
each cycle: replication of the original template DNA
and the 'long products' increases only linearly. The final PCR product is almost
entirely the unit length product.
That is, exponential replication of the DNA
target begins only of the fourth PCR cycle.
Compare this with the simplified diagram, which
shows the process as though it works beginning in the first
round of amplification.
Figure & text
©2024 by Steven M. Carr