Essential Cell Biology 5th edition

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DNA Cloning by PCR
341
DNA double helices
HEAT
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But these relatively weak, noncovalent bonds can be fairly easily broken—for
example, by heating the DNA to around 90ºC. Such treatment
will cause DNA denaturation, releasing the two strands from each other.
When the conditions are ECB5 reversed—by E10.04/10.10 slowly lowering the temperature—
the complementary strands will readily come back together to re-form a
double helix. This DNA renaturation, or hybridization, is driven by the
re-formation of the hydrogen bonds between complementary base pairs
(Figure 10–10).
Hybridization can be employed for detecting any nucleotide sequence
of interest, whether DNA or RNA. One simply designs a short, singlestranded
DNA probe that is complementary to that sequence. Because the
nucleotide sequences of so many genomes are known—and are stored in
publicly accessible databases—designing such a probe is straightforward.
The desired probe can then be synthesized in the laboratory—usually by
a commercial organization or a centralized academic facility.
Hybridization with DNA probes has many uses in cell and molecular biology.
As we will see later in this chapter, for example, DNA probes that
carry a fluorescent or radioactive label can be used to detect complementary
RNA molecules in tissue preparations. But one of the most powerful
applications of hybridization is in the cloning of DNA by the polymerase
chain reaction, as we discuss next.
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denaturation to
single strands
(hydrogen bonds between
nucleotide pairs broken)
SLOWLY
COOL
renaturation restores
DNA double helices
(nucleotide pairs re-formed)
Figure 10–10 A molecule of DNA can
undergo denaturation and renaturation
(hybridization). For two single-stranded
molecules to hybridize, they must have
complementary nucleotide sequences that
allow base-pairing. In this example, the red
and orange strands are complementary to
each other, and the blue and green strands
are complementary to each other. Although
denaturation by heating is shown, DNA
can also be denatured by alkali treatment.
The 1961 discovery that single strands of
DNA could readily re-form a double helix
in this way was a big surprise to scientists.
Hybridization can also occur between
complementary strands of DNA and RNA or
between two RNAs.
QUESTION 10–3
Discuss the following statement:
“From the nucleotide sequence of
a cDNA clone, the complete amino
acid sequence of a protein can be
deduced by applying the genetic
code. Thus, protein biochemistry has
become superfluous because there
is nothing more that can be learned
by studying the protein.”
DNA CLONING BY PCR
Genomic and cDNA libraries were once the only route to gene cloning,
and they are still used for cloning very large genes and for sequencing
whole genomes. However, a powerful and versatile method for amplifying
DNA, known as the polymerase chain reaction (PCR), provides
a more rapid and straightforward approach, particularly in organisms
whose complete genome sequence is known. Today, most genes are
cloned via PCR.
Invented in the 1980s, PCR revolutionized the way that DNA and RNA
are analyzed. The technique can amplify any nucleotide sequence
quickly and selectively. Unlike the traditional approach of cloning using
vectors—which relies on bacteria to make copies of the desired DNA
sequences—PCR is performed entirely in a test tube. Eliminating the
need for bacteria makes PCR convenient and fast—billions of copies of a
nucleotide sequence can be generated in a matter of hours. At the same
time, PCR is remarkably sensitive: the method can be used to amplify and
detect the trace amounts of DNA in a drop of blood left at a crime scene
or in a few copies of a viral genome in a patient’s blood sample. Because
of its sensitivity, speed, and ease of use, PCR has many applications in
addition to DNA cloning, including forensics and diagnostics.
In this section, we provide a brief overview of how PCR works and how
it is used for a range of purposes that require the amplification of specific
DNA sequences.