DISSERTATION

______________________________________________________________________ Introduction
1.3.2 Techniques for DNA microarray fabrication
A practical way of detecting and diagnosing various diseases is through the detection of nucleic
acid sequences, specific for any living organism 49 . One of the possible ways to gain insight into
a DNA sequence is by using DNA sensors. The sensitivity and selectivity of DNA biosensors
is highly dependent on the quality of the prepared DNA sensing surface. Good immobilization
technique ensures high reactivity, appropriate orientation, accessibility and the stability of the
grafted probe DNA and prevent unspecific binding 47 .
DNA immobilization methods can be divided into two groups 47,50 :
- Base-by-base synthesis (light-directed synthesis), which represents a bottom-up synthesis
of DNA sequences at the surface
- Direct attachment of already synthesized DNA sequences to the surface
Both strategies are used for DNA microarray fabrication. Several base-by-base synthesis
strategies were developed, including light-directed synthesis using photolithographic masks by
Affymetrix (GeneChips), photo-mediated synthesis by Roche (NimbleGen) that used digital
masks instead, and inkjet base-by-base manufacturing (printing) of DNA probes on the surface
developed by Agilent Technologies 51 . Among these, the Affymetrix strategy is the most known
and it will be shortly described here. On the other hand, among strategies for DNA array
fabrication by direct attachment of pre-synthesized DNA sequences, spotting (printing) is by
far the most known approach. However, new technologies for DNA array production are arising
on the market, including “electronic microarrays”.
Light-directed synthesis is a complex method requiring specialized equipment, however
attractive for DNA microarray fabrication. The principle of in situ DNA synthesis by
Affymetrix consists of UV masking and light-directed chemical synthesis of DNA sequences
directly at the array, one nucleotide at the time per spot for many spots simultaneously (Figure
1.10). The surface is initially modified with a covalent linker containing a protection group that
is easily removed upon irradiation. Using a photolithographic mask, desired spots on the surface
are irradiated, removing locally the protecting groups. Subsequently, these spots are modified
with the desired protected nucleotides. This experimental sequence is repeated as many times
as necessary to obtain the desired DNA sequences on the surface and this is determined by the
1.3 DNA immobilization 18