Essential Cell Biology 5th edition

342 CHAPTER 10 Analyzing the Structure and Function of Genes
PCR Uses DNA Polymerase and Specific DNA Primers to
Amplify DNA Sequences in a Test Tube
The success of PCR depends on the exquisite selectivity of DNA hybridization,
along with the ability of DNA polymerase to copy a DNA template
reliably, through repeated rounds of replication in vitro. The enzyme
works by adding nucleotides to the 3′ end of a growing strand of DNA (see
Figure 6−11). To initiate the reaction, the polymerase requires a primer—
a short nucleotide sequence that provides a 3′ end from which synthesis
can begin. The beauty of PCR is that the primers that are added to the
reaction mixture not only serve as starting points, but they also direct the
polymerase to the specific DNA sequence to be amplified. These primers
are designed by the experimenter based on the DNA sequence of interest
and then synthesized chemically. Thus, PCR can only be used to clone a
DNA segment for which the sequence is known in advance. However,
with the large and growing number of genome sequences available in
public databases, this requirement is rarely a drawback.
The power of PCR comes from repetition: the cycle of amplification is
carried out dozens of times over the course of a few hours. At the start
of each cycle, the two strands of the double-stranded DNA template
are separated and a unique primer is hybridized, or annealed, to each.
DNA polymerase is then allowed to replicate each strand independently
(Figure 10–11). In subsequent cycles, all the newly synthesized DNA molecules
produced by the polymerase serve as templates for the next round
of replication (Figure 10–12). Through this iterative process of amplification,
many copies of the original sequence can be made—billions after
about 20 to 30 cycles.
PCR is the method of choice for cloning relatively short DNA fragments
(say, under 10,000 nucleotide pairs). Because the original template for
PCR can be either DNA or RNA, the method can be used to obtain either
a full genomic clone (complete with introns and exons) or a cDNA copy
of an mRNA (Figure 10–13). A major benefit of PCR is that genes can be
cloned directly from any piece of DNA or RNA without the time and effort
needed to first construct a DNA library.
5′
3′
region of
double-stranded
DNA to be
amplified
3′
5′
STEP 1
HEAT TO
SEPARATE
STRANDS
STEP 2
COOL TO
ANNEAL
PRIMERS
STEP 3
DNA SYNTHESIS
+ DNA polymerase
+ dATP
+ dGTP
+ dCTP
+ dTTP
5′ 3′
3′
5′
5′
3′
3′
5′
products of
first cycle
pair of
primers
FIRST CYCLE OF AMPLIFICATION
Figure 10–11 A pair of PCR primers directs the amplification of a desired segment of DNA in a test tube. Each cycle of PCR
includes three steps: (1) The double-stranded DNA is heated briefly to separate the two strands. (2) The DNA is exposed to a large
excess of a pair of specific primers—designed to bracket the region of DNA to be amplified—and the sample is cooled to allow the
primers to hybridize to complementary sequences in the two DNA strands. (3) This mixture is incubated with DNA polymerase and
the four deoxyribonucleoside triphosphates so that DNA can be synthesized, starting from the two primers. The process can then be
repeated by reheating the sample to separate the double-stranded products of the previous cycle (see Figure 10−12).
The technique depends on the use of a special DNA polymerase isolated from a thermophilic bacterium; this polymerase is stable at
much higher temperatures than eukaryotic DNA polymerases, so it is not denatured by the heat treatment shown in step 1. The enzyme
therefore does not have to be added again after each cycle.
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