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Chapter 4
4.1 Introduction
Single nucleotide polymorphisms (SNPs) are one of major sources of genetic variation in the
human genome 1 . Many SNPs have been linked to several diseases or pathological conditions,
such as the Alzheimer disease (AD), which was found to be related to SNPs located in the
ApoE gene. This gene encodes for the apolipoprotein E, a protein involved in cholesterol
metabolism. This protein has been found to exist in three main isoforms (ε2, ε3, ε4) 2 that
differ for the substitution of amino acids at position 112 and 158: Cys112/Cys158 (ε2),
Cys112/Arg158 (ε3) and Arg112/Arg158 (ε4). The combination of the variants on the two
alleles generates six different genotypes 3 . An increased frequency of the ApoE ε4 allele in
late-onset AD patients, both familiar and sporadic, has been demonstrated, and a gene dose
effect for the ε4 allele was found in AD families, implicating that this allele is a major risk
factor for AD 4 .
Selective SNP recognition is useful both in diagnostic and therapeutic applications. For
example, in the case of AD, having this disease origin in early-life 5 and given that no cures
are at the moment available, accurate and early diagnosis of a genetic predisposition could be
very important, since early therapeutic intervention, before severe cellular damages, would
improve the prognosis and life quality of AD patients. Moreover, since AD diagnosis can be
confirmed only by autoptic post-mortem analysis, a reliable genetic testing to be performed
on all subjects suffering for dementia might help to distinguish between AD and non-AD
subjects 6 .
Recently, great efforts have been devoted to develop selective and specific methods for SNP
analysis, yielding a large number of distinct approaches 7 . Usually, a PCR amplification of the
desired SNP-containing region is initially performed in order to specifically increase the
number of DNA molecules for detection. After PCR, allelic discrimination is done by
different strategies: primer extension (nucleotide incorporation), ligation, enzymatic cleavage
and hybridization 7 . Hybridization approaches exploit differences in thermal stability of
double-stranded DNA to distinguish between matched and mismatched target-probe pairs.
Thus, it is critical to perform assays under conditions where hybridization occurs only if the
probe and the target are perfectly complementary.
Selective hybridization methods have been implemented not only in solution but also on
surface-based methods, such as microarrays 8,9 . Such methods usually rely on the recognition
of a DNA target by hybridization with a single strand oligonucleotide probe immobilized onto
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