KEY TERMS:
- Denaturation of protein describes its conversion from the physiological conformation to some other (inactive) conformation.
- Renaturation describes the reassociation of denatured complementary single strands of a DNA double helix.
- Annealing of DNA describes the renaturation of a duplex structure from single strands that were obtained by denaturing duplex DNA.
- Hybridization describes the pairing of complementary RNA and DNA strands to give an RNA-DNA hybrid.
- Heating causes the two strands of a DNA duplex to separate.
- The Tm is the midpoint of the temperature range for denaturation.
- Complementary single strands can renature when the temperature is reduced.
- Denaturation and renaturation/hybridization can occur with DNA-DNA, DNA-RNA, or RNA-RNA combinations, and can be intermolecular or intramolecular.
- The ability of two single-stranded nucleic acid preparations to hybridize is a measure of their complementarity.
The concept of base pairing is central to all processes
involving nucleic acids. Disruption of the base pairs is a crucial aspect of the
function of a double-stranded molecule, while the ability to form base pairs is
essential for the activity of a single-stranded nucleic acid.Figure 1.16 shows that base pairing enables complementary
single-stranded nucleic acids to form a duplex structure.
- An intramolecular duplex region can form by base pairing between two complementary sequences that are part of a single-stranded molecule.
- A single-stranded molecule may base pair with an independent, complementary single-stranded molecule to form an intermolecular duplex.
Formation of duplex regions from single-stranded nucleic
acids is most important for RNA, but single-stranded DNA also exists (in the
form of viral genomes). Base pairing between independent complementary single
strands is not restricted to DNA-DNA or RNA-RNA, but can also occur between a
DNA molecule and an RNA molecule.
The lack of covalent links between complementary strands
makes it possible to manipulate DNA in vitro. The noncovalent forces
that stabilize the double helix are disrupted by heating or by exposure to low
salt concentration. The two strands of a double helix separate entirely when all
the hydrogen bonds between them are broken.
The process of strand separation is called denaturation or (more colloquially) melting.
("Denaturation" is also used to describe loss of authentic protein structure; it
is a general term implying that the natural conformation of a macromolecule has
been converted to some other form.)
Denaturation of DNA occurs over a narrow temperature range
and results in striking changes in many of its physical properties. The midpoint
of the temperature range over which the strands of DNA separate is called the
melting temperature (Tm). It depends on the
proportion of G·C base pairs. Because each G·C base pair has three hydrogen bonds, it is more stable
than an A·T base pair, which has only two hydrogen
bonds. The more G·C base pairs are contained in a
DNA, the greater the energy that is needed to separate the two strands. In
solution under physiological conditions, a DNA that is 40% G·C—a value typical of
mammalian genomes—denatures with a
Tm of about 87°C. So duplex
DNA is stable at the temperature prevailing in the cell.
The denaturation of DNA is reversible under appropriate
conditions. The ability of the two separated complementary strands to reform
into a double helix is called renaturation.
Renaturation depends on specific base pairing between the complementary strands.
Figure 1.17 shows that the reaction takes place in two
stages. First, single strands of DNA in the solution encounter one another by
chance; if their sequences are complementary, the two strands base pair to
generate a short double-helical region. Then the region of base pairing extends
along the molecule by a zipper-like effect to form a lengthy duplex molecule.
Renaturation of the double helix restores the original properties that were lost
when the DNA was denatured.
Renaturation describes the reaction between two
complementary sequences that were separated by denaturation. However, the
technique can be extended to allow any two complementary nucleic acid sequences
to react with each other to form a duplex structure. This is sometimes called
annealing, but the reaction is more generally
described as hybridization whenever nucleic acids
of different sources are involved, as in the case when one preparation consists
of DNA and the other consists of RNA. The ability of two nucleic acid
preparations to hybridize constitutes a precise test for their complementarity
since only complementary sequences can form a duplex
structure.
The principle of the hybridization reaction is to expose two
single-stranded nucleic acid preparations to each other and then to measure the
amount of double-stranded material that forms. Figure 1.18
illustrates a procedure in which a DNA preparation is denatured and the single
strands are adsorbed to a filter. Then a second denatured DNA (or RNA)
preparation is added. The filter is treated so that the second preparation can
adsorb to it only if it is able to base pair with the DNA that was originally
adsorbed. Usually the second preparation is radioactively labeled, so that the
reaction can be measured as the amount of radioactive label retained by the
filter.
The extent of hybridization between two single-stranded
nucleic acids is determined by their complementarity. Two sequences need not be
perfectly complementary to hybridize. If they are closely related but
not identical, an imperfect duplex is formed in which base pairing is
interrupted at positions where the two single strands do not correspond.
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