Mutational analysis of large genes with complex genomic structures plays an important role in medical genetics. Technical limitations associated with current mutation screening protocols have placed increased emphasis on the development of new technologies to simplify these procedures. High-density arrays of >90,000-oligonucleotide probes, 25 nucleotides in length, were designed to screen for all possible heterozygous germ-line mutations in the 9.17-kb coding region of the ATM gene. A strategy for rapidly developing multiexon PCR amplification protocols in DNA chip-based hybridization analysis was devised and implemented in preparing target for the 62 ATM coding exons. Improved algorithms for interpreting data from two-color experiments, where reference and test samples are cohybridized to the arrays, were developed. In a blinded study, 17 of 18 distinct heterozygous and 8 of 8 distinct homozygous sequence variants in the assayed region were detected accurately along with five false-positive calls while scanning >200 kb in 22 genomic DNA samples. Of eight heterozygous sequence changes found in more than one sample, six were detected in all cases. Five previously unreported sequence changes, not found by other mutational scanning methodologies on these same samples, were detected that led to either amino acid changes or premature truncation of the ATM protein. DNA chip-based assays should play a valuable role in high throughput sequence analysis of complex genes.

Pairs of high density oligonucleotide arrays (DNA chips) consisting of >96 000 oligonucleotides were designed to screen the entire 5.53 kb coding region of the hereditary breast and ovarian cancer BRCA1 gene for all possible sequence changes in the homozygous and heterozygous states. Single-stranded RNA targets were generated by PCR amplification of individual BRCA1 exons using primers containing T3 and T7RNA polymerase promoter tails followed by in vitro transcription and partial fragmentation reactions. Fluorescent hybridization signals from targets containing the four natural bases to >5592 different fully complementary 25mer oligonucleotide probes on the chip varied over two orders of magnitude. To examine the thermodynamic contribution of rU.dA and rA.dT target.probe base pairs to this variability, modified uridine [5-methyluridine and 5-(1-propynyl)-uridine)] and modified adenosine (2,6-diaminopurine riboside) 5'-triphosphates were incorporated into BRCA1 targets. Hybridization specificity was assessed based upon hybridization signals from >33 200 probes containing centrally localized single base pair mismatches relative to target sequence. Targets containing 5-methyluridine displayed promising localized enhancements in hybridization signal, especially in pyrimidine-rich target tracts, while maintaining single nucleotide mismatch hybridization specificities comparable with those of unmodified targets.

The energies of plectonemic and toroidal supercoiled DNA are calculated by treating DNA as an elastic rod with a finite radius. End effects are ignored and all extensive quantities (e.g., writhe, bend energy) are treated as linear densities (writhe per unit length, bend energy per unit length). Minimum energy configurations are found. For plectonemic DNA, the superhelical pitch angle cz is in the range 45" < ag90". For low values of specific linking difference, most superhelicity is in writhe. As specific linking difference increases, a greater proportion of superhelicity is in twist. Under physiological conditions, roughly 8X% of superhelicity is in writhe. Ionic strength effects are discussed, and it is found that variation of excluded volume with ionic strength has a large effect, resulting in significantly greater torsional stress in supercoiled DNA at low ionic strength. For biologically relevant values of specific linking difference, the plectonemic conformation is energetically favored over toroidal conformations. Results are compared with electron microscopy data. The application of the model to DNA conformational transitions is discussed. strength.45A8 J. Chem. Phys. 95 (12), 15 December 1991

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