C. Prinsen, K.Hardy, F.L. van der Loop#, H.J.M. Smeets#, B. Holloway*, F.B.J.M Thunnissen.
Departments of Pathology, Canisius-Wilhelmina Ziekenhuis, Nijmegen and #Genetics, Maastricht University, Maastricht, the Netherlands and *Center for Disease Control, Atlanta, USA.
First generation microarrays are characterized by hybridization as capture approach and
subsequent detection of the label. Single or mixed dual fluorescence labels of (c)DNA or
RNA are used for visualization and analyzes. With this conventional approach the samples
to be tested still need to contain >20-50% informative cells (target fraction), which
is in clinical samples infrequently the case. Recently, an application of a highly
sensitive microtiter based point mutation analysis technique has been published requiring
a target fraction of 0.01% (Point-EXACCT, J Clin. Oncol. 1998, 16, 3061-3068). The purpose
of the present study was to transfer that technique to microarray format.
The results show that biotin labeled probes can
be spotted to glass slides for capture function. After serial hybridization firstly with
unlabeled target DNA and then with labeled detection probe, both probes can be ligated
together. The presence of the detection probe was visualized after alkaline phosphatase
immunostaining with absorption light microscopy. Mutation analysis has been validated with
the results of the microtiter plate format (n=10 cases). This microarray procedure is
sensitive, rapid, non-radioactive and can easily be automated. In addition, more mutations
can be analysed in the same run. Since the microtiter format was suitable for mutation
detection in sputum the present microarray analyses offers perspectives for use in
specimen with low target fractions.
In conclusion, a detection approach is described allowing easy visual examination of a large number of clinical samples for mutation and SNP analyses.