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One solution for increasing the number of different targets that are amplifi ed in
a single PCR reaction is the spatial separation of different primer pairs. Microarrays
are ideally suited for this task: tethering each pair of primers to a discrete spot on a
surface directs the amplifi cation of different targets in a number of non-overlapping
micro-surroundings. Given the miniature dimensions of microarrays, highly multi-
plexed amplifi cation would likewise occur in a homogenous, minimal volume and
avoid the split assay. In multiplex microarray-enhanced PCR (MME-PCR), a
highly sensitive method for DNA analysis, two amplifi cation strategies are carried
out simultaneously: on or within gel elements, and in bulk solution over the gel
element array.
A universal DNA microarray combining PCR and ligase detection reaction
(LDR) exploits full use of the sensitivity that the ligase detection reaction can pro-
vide, while maintaining a rapid read out on a universal microarray. It becomes fully
programmable by uncoupling the mutation detection step from array hybridization.
Main features of this method are:
- After hybridization of a discriminating probe and a common probe to the target
sequence, ligation occurs only if there is perfect complementarity between the
two probes and the template. - The reaction is thermally cycled, generating single-stranded DNA fragments
bearing a 5′ Cy3 fl uorescent moiety and a 3′ czip code sequence. - The cycling enables a more common probe (and the corresponding czip code) to
ligate to the discriminating probe, given a fi xed amount of PCR target. - The LDR product is hybridized to a universal microarray, where unique zip code
sequences have been spotted.
PCR/LDR/universal DNA microarray is 50-fold more sensitive and 10-fold more
rapid than conventional hybridization-only arrays. It is a promising technology to
help drive the transition from the current paradigms of clinical decision making to
the new era of personalized medicine.
Gene Profi ling Array
A Gene Profi ling Array (Affymetrix) is made using spatially patterned, light-
directed combinatorial chemical synthesis and contain up thousands of different
oligonucleotides on a small glass surface. In this approach sequence information is
used directly to design high-density, 2D arrays of synthetic oligonucleotides, which
are used for quantitative and highly parallel measurements of gene expression, to
discover polymorphic loci and to detect the presence of thousands of alternative
alleles. The latest product, Affymetrix® Gene Profi ling Array cGMP U133 P2,
gives the protein-coding content of the human genome on a single piece the size of
a fi ngernail.
2 Molecular Diagnostics in Personalized Medicine