94 ANALYSIS OF DNA SEQUENCES BY HYBRIDIZATION
Figure 3.24 |
Three ligands used in stoichiometric amplification systems. |
(a) Digoxigenin. |
(b) |
Biotin. (c)Fluorescein. All are shown as derivatives of dpppU, but other derivatives are available.
bodies |
and |
their |
complexes |
with |
haptens |
are |
reasonably |
stable, |
streptavidin- |
biotin |
complexes are |
much more stable and |
can survive extremes of temperature and |
pH |
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in ways comparable to DNA. Parenthetically, a disadvantage of the streptavidin system is
that the protein and its complexes are so stable that it is very difficult to reverse them to generate free DNA again, if this is needed. Even greater degrees of signal amplification can be achieved by using dendrimers as described in Box 3.6.
All of these amplification systems work well, but they do not have the same power of sample multiplication that can be achieved when the amplification is carried out directly at the DNA level by enzymatic reactions. Such methods are the subject of the next chapter. Ultimately sample amplification systems can be combined with color-generating amplification systems to produce exquisitely sensitive ways of detecting multiple DNA samples, sometimes in multiple colors.
Figure |
3.25 |
The three-dimen- |
|
sional |
structure of |
streptavidin. |
|
Four bound biotin molecules are |
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shown |
in |
boldface. |
(Illustration |
created by Sandor Vajda using |
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protein |
coordinates |
provided by |
|
Wayne Hendrickson.)
Figure 3.26 |
Detailed structural intermediates formed by two methods for stoichiometric amplifi- |
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cation. |
(a)Streptavidin and some other protein containing multiple attached biotin (b) residues. |
(b) |
|
A monoclonal antibody directed against fluorescein (F) and a fluorescinated polyclonal antibody |
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specific for the monoclonal antibody. |
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96 ANALYSIS OF DNA SEQUENCES BY HYBRIDIZATION
BOX 3.6
DENDRIMERIC DNA PROBES
Dendrimers are a chemical amplification system that allows large structures to be constructed by systematic elaboration of smaller ones. A traditional dendrimer is formed by successive covalent additions of branched reactive species to a starting framework.
Each layer added grows the overall mass of |
the structure |
considerably. The |
process is |
||
a polyvalent |
analogue of |
the stoichiometric |
amplification |
schemes described |
in Fig- |
ure 3.26. |
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|
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|
Recently schemes have been designed and implemented to construct dendrimeric |
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arrays of DNA |
molecules. |
Here branched structures are used to create polyvalency, |
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and base-pairing specificity is used to direct the addition of each successive layer. The types of structures used and the complexity of the products that can be formed are illustrated schematically in Figure 3.27. These structures are designed so that each layer presents equal amounts of two types of single-stranded arms for further complexation.
Ultimately one |
type of arm is used |
to identify a specific target by base pairing, while |
the other type |
of arm is used to |
bind molecules needed for detection. Dendrimers |
could be built on a target layer by |
layer, or they can be preformed with specificity se- |
|
lected for each particular target of interest. The latter approach appears to offer a major increase in sensitivity in a range of biological applications including Southern blots, and in situ hybridization.
Figure 3.27 Dendrimer layer growth. Figure also appears in color insert. (Illustration provided by Thor Nilsson.)
SOURCES AND ADDITIONAL READINGS |
97 |
SOURCES |
AND ADDITIONAL |
READINGS |
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83: 3746–3750. |
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Cantor and |
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Chemistry |
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W. |
H. |
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