Protein microchips: use for immunoassay and enzymatic reactions.

Different proteins such as antibodies, antigens, and enzymes were immobilized within the 100 x 100 x 20-microm gel pads of protein microchips. A modified polyacrylamide gel has been developed to accommodate proteins of a size up to 400,000 daltons. Electrophoresis in the microchip reaction chamber speeded up antigen-antibody interactions within the gel. Protein microchips were used in immunoassays for detection of antigens or antibodies, as well as to carry out enzymatic reactions and to measure their kinetics in the absence or presence of an inhibitor. A protein microchip can be used several times in different immunoassays and enzymatic kinetic measurements.

Manual manufacturing of oligonucleotide, DNA, and protein microchips.

A simple procedure for manufacturing microchips containing various gel-immobilized compounds is described. A gel photopolymerization technique is introduced to produce micromatrices of polyacrylamide gel pads (25 x 25 x 20 microm and larger) separated by a hydrophobic glass surface. A pin device for the manual application of a compound in solution onto the activated polyacrylamide gel pad for immobilization is described. Oligonucleotide, DNA, and protein microchips have been produced by this method and tested by hybridization and immunoanalysis monitored with a fluorescence microscope. The effect of the lengths of the immobilized oligonucleotides and the hybridized RNA and DNA on hybridization of the oligonucleotide microchips was evaluated. This method can also be used for manufacturing microchips containing a variety of other compounds.

DNA sequence recognition by hybridization to short oligomers: experimental verification of the method on the E. coli genome.

A newly developed method for sequence recognition by hybridization to short oligomers is verified for the first time in genome-scale experiments. The experiments involved hybridization of 15,328 randomly selected 2-kb genomic clones of Escherichia coli with 997 short oligomer probes to detect complementary oligomers within the clones. Lists of oligomers detected within individual clones were compiled into a database. The database was then searched using known E. coli sequences as queries. The goal was to recognize the clones that are identical or similar to the query sequences. A total of 76 putative recognitions were tested in two separate but complementary recognition experiments. The results indicate high specificity of recognition. Current and prospective applications of this novel method are discussed.

Genome-scale DNA sequence recognition by hybridization to short oligomers.

Recently developed hybridization technology (Drmanac et al. 1994) enables economical large-scale detection of short oligomers within DNA fragments. The newly developed recognition method (Milosavljevic 1995b) enables comparison of lists of oligomers detected within DNA fragments against known DNA sequences. We here describe an experiment involving a set of 4,513 distinct genomic E.coli clones of average length 2kb, each hybridized with 636 randomly selected short oligomer probes. High hybridization signal with a particular probe was used as an indication of the presence of a complementary oligomer in the particular clone. For each clone, a list of oligomers with highest hybridization signals was compiled. The database consisting of 4,513 oligomer lists was then searched using known E.coli sequences as queries in an attempt to identify the clones that match the query sequence. Out of a total of 11 clones that were recognized at highest significance level by our method, 8 were single-pass sequenced from both ends. The single-pass sequenced ends were then compared against the query sequences. The sequence comparisons confirmed 7 out of the total of 8 examined recognitions. This experiment represents the first successful example of genome-scale sequence recognition based on hybridization data.

Sequencing by hybridization: towards an automated sequencing of one million M13 clones arrayed on membranes.

An immediately applicable variant of the sequencing by hybridization (SBH) method is under development with the capacity to determine up to 100 million base pairs per year. The proposed method comprises six steps: (i) arraying genomic or cDNA M13 clones in 864-well plates (wells of 2 mm); (ii) preparation of DNA samples for spotting by growth of the M13 clones or by polymerase chain reaction (PCR) of the inserts using standard 96-well plates, or plates having as many as 864 correspondingly smaller wells; (iii) robotic spotting of 13,824 samples on an 8 x 12 cm nylon membrane, or correspondingly more, on up to 6 times larger filters, by offset printing with a 96 or 864 0.4 mm pin device; (iv) hybridization of dotted samples with 200-2000 32P-labeled probes comprising 16-256 10-mers having a common 8-mer, 7-mer, or 6-mer in the middle (20 probes per day, each hybridized with 250,000 dots); (v) scoring hybridization signals of 5 million sample-probe pairs per day using storage phosphor plates; and (vi) computing clone order and partial-to-complete DNA sequences using various heuristic algorithms. Genome sequencing based on a combination of this method and gel sequencing techniques may be significantly more economical than gel methods alone.

Global approaches to quantitative analysis of gene-expression patterns observed by use of two-dimensional gel electrophoresis.

A major difficulty in the use of two-dimensional protein maps to identify and classify cell types is the problem of acquiring, selecting, and analyzing quantitative data on hundreds of protein spots. Here we use methods of multivariate statistics to analyze the differences among a panel of human cell lines, in some cases involving quantitative data on more than 250 proteins. Principal-component and cluster-analysis techniques show that the lines can be easily distinguished, even by using the subset of proteins present in all cells. A preliminary analysis of the protein changes brought about by phorbol ester-induced differentiation of the line U937 is included.