Essential Cell Biology 5th edition

DNA Cloning by PCR
343
HEAT TO
SEPARATE
STRANDS
AND
COOL TO
ANNEAL
PRIMERS
DNA
SYNTHESIS
HEAT TO
SEPARATE
STRANDS
AND
COOL TO
ANNEAL
PRIMERS
DNA
SYNTHESIS
products of
first cycle
END OF
FIRST CYCLE
SECOND CYCLE
(produces four double-stranded
DNA molecules)
THIRD CYCLE
(produces eight double-stranded
DNA molecules)
Figure 10–12 PCR uses repeated rounds of strand separation, hybridization, and synthesis to amplify DNA. As
the procedure outlined in Figure 10–11 is repeated, all the newly synthesized fragments serve as templates in their
turn. Because the polymerase and the primers remain in the sample after the first cycle, PCR involves simply heating
and then cooling the same sample, in the same test tube, again and again. Each cycle doubles the amount of DNA
ECB5 e10.15/10.12
synthesized in the previous cycle, so that within a few cycles, the predominant DNA is identical to the sequence
bracketed by and including the two primers in the original template. In the example illustrated here, three cycles
of reaction produce 16 DNA chains, 8 of which (boxed in yellow) correspond exactly to one or the other strand of
the original bracketed sequence. After four more cycles, 240 of the 256 DNA chains will correspond exactly to the
original sequence, and after several more cycles, essentially all of the DNA strands will be this length. The whole
procedure is shown in Movie 10.1.
QUESTION 10–4
PCR Can Be Used for Diagnostic and Forensic
Applications
In addition to its use in cloning, PCR is frequently employed to amplify
DNA for other, more practical purposes. Because of its extraordinary sensitivity,
PCR can be used to detect an infection at its earliest stages. In
this case, short sequences complementary to the suspected pathogen’s
genome are used as primers, and following many cycles of amplification,
even a few copies of an invading bacterial or viral genome in a
patient sample can be detected (Figure 10–14). PCR can also be used to
track epidemics, detect bioterrorist attacks, and test food products for
the presence of potentially harmful microbes. It is also used to verify the
authenticity of a food source—for example, whether a sample of beef
actually came from a cow.
Finally, PCR is widely used in forensic medicine. The method’s extreme
sensitivity allows forensic investigators to isolate DNA from even the
smallest traces of human blood or other tissue to obtain a DNA fingerprint
of the person who left the sample behind. With the possible exception
of identical twins, the genome of each human differs in DNA sequence
from that of every other person on Earth. Using primer pairs targeted at
genome sequences that are known to be highly variable in the human
A. If the PCR shown in Figure
10–12 is carried through an
additional two rounds of
amplification, how many of the
DNA fragments (gray, green,
red, or outlined in yellow) will be
produced? If many additional cycles
are carried out, which fragments will
predominate?
B. Assume you start with one
double-stranded DNA molecule
and amplify a 500-nucleotide-pair
sequence contained within it.
Approximately how many cycles
of PCR amplification will you need
to produce 100 ng of this DNA?
100 ng is an amount that can be
easily detected after staining with
a fluorescent dye. (Hint: for this
calculation, you need to know that
each nucleotide has an average
molecular mass of 330 g/mole.)