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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.

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.

Liver proteins of male C57BL/6T mice treated with 0, 50, or 250 mg/kg Aroclor 1 254 were analyzed by high-resolution two-dimensional (2-D) electrophoresis. The resulting patterns were processed using a computerized image analysis system and quantitative data selected for a total of 150 protein spots. On the basis of an analysis of liver proteins from five animals in each treatment group, we found 3 1 proteins that showed quantitative differences attributable to treatment with chlorinated hydrocarbons at a high level of statistical significance. One ofthe altered proteins appears to be Mitcon:2, a heat-shock sensitive mitochondria1 matrix polypeptide; another appears likely to be microsomal cytochrome b5. The results indicate that quantitative 2-D protein mapping may reveal much more detail regarding the in vivo effects of toxic xenobiotics than has previously been available, and thus allow a more informative approach to the testing of toxic compounds.