Affymetrix GeneChip hybridization system has become widely used standard for expression genomics investigations because it offers both high reproducibility and sensitivity when analyzing extremely large number of genes simultaneously. Nevertheless, because processing expenses are quite high and detection limits are often obstacle, it presents irresistible challenges for modifying standard protocols in achieving better target quality and higher detection sensitivity at lower cost. We have modified and optimized more than 80% of the original methodology and created a set of customized protocols and kits. These lead to approximately 50% savings in processing reagents and time while significantly increasing enzymatic reaction yields (5-10-fold) and minimizing detection limits (10-100-fold). Every single modification was carefully monitored and compared to the original Affymetrix-recommended protocol before being adopted as a standard procedure - only when proven to deliver significant (at least 2-fold) advantage over the originally proposed.

H.M. Dyanov, D. Salbego, S. Savcovic, H. Kreuzer, H. Serrato, S. Batus, D. Grujic, M. Zeremski, Z. Strezovska, T. Paunesku, S. Little, M. Koutrev and P. Dyanova

This successful strategy was developed, tested and presented in 1995 for large-scale cDNA library clustering and characterization based on oligonucleotide hybridization with more than 200 oligonucleotide probes and computerized data analysis. It creates oligonucleotide “fingerprints” allowing unique identification of a cDNA clone from many thousands other clones. Our proof-of-concept methodology was designated to test the ability of the approach to provide gene identification fingerprints, comprehensive catalogues of genes and to reveal gene expression patterns. Our final results by using only 217 computationally designed probes demonstrated very precise clustering – all of the analyzed cDNA clusters contained cDNA clones, which corresponded to the same or a highly similar cDNA/mRNA type some of which were identified by GenBank similarity search. Briefly, two research teams have previously separated about 132 000 cDNA clones from infant brain cDNA libraries, have amplified their inserts by PCR, and arrayed them onto GeneScreen™ nylon membranes. cDNAs were hybridized with oligonucleotides at miss-match eliminating conditions and clustered computationally based on their hybridization signatures.

The DNA sequencing by hybridization (SBH) Format-1 technique (Drmanac, R. et al., 1993) is based on massive DOT-hybridization experiments with short oligomer probes, consecutively hybridized with arrays of clones fixed on nylon membrane. A dot-hybridization was used to define a presence of unique oligonucleotide sequence signatures in corresponding arrayed clones. In our experimental design each probe consist of 33P-dNTP-labeled oligomer of defined formula (N)0-2(oligo)6-11(N)0-2, hybridized in miss-matches eliminating conditions to the 500-2,500 bp PCR-fragments as nylon-arrayed targets. The presented here technique, developed in 1995, is a highly productive and optimally designed procedure for large-scale recombinant cDNA-clones collection, storage, growth, PCR-amplification, dot-spotting, hybridization, phosphor-image creating, and sequencing data generation; most of them robotically performed.