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288 Edwards and Bartlett
technology over radioactive are (1) an internal molecular weight standard is used in each
lane to align data, thereby eliminating lane-to-lane variability; (2) strands of DNA in the
PCR amplicon can be labeled with different-colored labels; And (3) there is the ability
to multiplex products from different PCRs in one lane to increase sample throughput.
SSCP is further discussed in Chapter 48. However, although SSCP is a useful screening
technique, it yields no information on the location of the mutation. A band shift will
identify the presence of the mutation within the PCR product, but no sequence information
on the exact base that has been mutated can be determined without sequencing.
2.1.2. Mutational Detection Using Cleavage Systems
2.1.2.1. GEL-BASED METHOD
An alternative screening method for mutations or polymorphisms that does provide
some information on the location of the mutation is enzymatic cleavage of DNA.
Enzymatic cleavage systems rely on enzymes such as resolvases (e.g., the enzyme T4
endonuclease VII; ref. 4). These enzymes cleave double-stranded DNA at sites where
a “bubble” is formed because of miss-pairing of bases. Mutation analysis is performed
by mixing PCR products from the test samples and samples with known sequences
(either normal alleles or wild type DNA). The PCR products are melted and the DNA
strands allowed to re-anneal. In the presence of either a mutation or a different allele
from the wild type, a miss-match in sequence provides a target for the resolvase
enzyme. The enzyme scans along the double-stranded DNA and binds to the singlestranded
“bubble” at the site of the miss-match, and the DNA is then cleaved in
this region. The resultant fragments are then separated by electrophoresis, and the
presence of cleavage products indicates the presence of a mutation (4,5). The size of
the cleaved fragments indicates the approximate location of the mutation, which is
an advantage over SSCP.
2.1.2.2. NONGEL-BASED METHOD
The method just discussed uses a gel-based detection system. An example of a
cleavage method that is not gel based is the use of a structure-specific 5′ nuclease to
cleave sequence-specific structures in each of two cascading reactions (6). The cleavage
structure forms when two synthetic oligonucleotides hybridize in tandem to a target.
One of the oligonucleotides cycles on and off the target and is cut by the nuclease
only when the appropriate structure forms. The cleaved probes then participate in a
second reaction involving a dye-labeled fluorescence resonance energy-transfer probe.
Cleavage of this probe generates a signal, which can be analyzed by fluorescence
microtiter plate readers (6).
2.1.3. Oligonucleotide Microarrays
Current screening methods are rapid and when combined with gel-based fluorescent
DNA sequencing technology can accurately locate and identify mutations and polymorphisms.
However, SNPs are now being uncovered and assembled into large SNP
databases. Although this will be invaluable for linking SNPs with disease, it will require
analysis of thousands of polymorphisms (7). Currently, there is no technique available
that is fast, specific, sensitive, reliable, and cost-effective enough for genome-wide
polymorphism analysis. Oligonucleotide microarrays are currently under intensive