A simple and rapid method for the analysis of genetic polymorphisms has been developed using allele-specific oligonucleotide arrays bound to glass supports. Allele-specific oligonucleotides are covalently immobilized on glass slides in arrays of 3 mm spots. Genomic DNA is amplified by PCR using one fluorescently tagged primer oligonucleotide and one biotinylated primer oligonucleotide. The two complementary DNA strands are separated, the fluorescently tagged strand is hybridized to the support-bound oligonucleotide array, and the hybridization pattern is detected by fluorescence scanning. Multiple polymorphisms present in the PCR product may be detected in parallel. The effect of spacer length, surface density and hybridization conditions were evaluated, as was the relative efficacy of hybridization with single or double-stranded PCR products. The utility of the method was demonstrated in the parallel analysis of 5 point mutations from exon 4 of the human tyrosinase gene.

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Plasmids containing the VA RNA genes of adenovirus are faithfully transcribed by a crude cytoplasmic extract containing DNA-dependent RNApolymerase II [Wu, G.-J. (1978) Proc. NatI. Acad. Sci. USA 75, 2175USA 75, -2179. By subjecting these DNA templates to in vitro site-directed mutagenesis with a novel enzyme of Pseudomonas and recloning in pBR322, we have constructed an ordered series of deletions which affect the in vitro transcription of the major RNA polymerase III viral product, VA, RNA. Three regions that are required for specific synthesis of VA, RNA can be defined. One, inside the gene at nucleotides + 10 to +76, affects the transcription in an all-or-none fashion. Transcription is initiated on plasmid sequences that replace up to 10 nucleotides downstream from the 5' end ofthe gene. The development of soluble cell-free extracts in which specific genes are selectively and accurately transcribed has greatly facilitated our understanding of the molecular basis of control in prokaryotes (1) and eukaryotes (2)(3)(4)(5)(6). Faithful in vitro transcription systems were first developed for RNA polymerase III products, like the adenovirus encoded 5.5S RNA (VA RNA) by Wu (2) and later modified (4) or the Xenopus 5S genes (3, 7). Appropriate templates in large amounts and with high purity can now be obtained by recombinant DNA techniques. These templates can be manipulated to determine the DNA signals required for control of accurate initiation or termination of transcription.Recently it was shown that, for the 5S genes ofXenopus laevis, a region internal to the gene was required for specific initiation of transcription mediated by oocyte cell extracts (8,9). A purified factor required for accurate initiation strongly bound to this region of the gene (10) and might participate in the temporal control of 5S transcription in vivo because it also binds to the 5S RNA gene product (10,11). A different set of factors seems to be required for adenovirus VA transcription (10) and it was therefore ofinterest to establish whether a similar internal control region could be identified for this RNA (15,16,21).Using the DNA sequence data from this region ofthe genome (17,18,(21)(22)(23) and appropriate restriction enzymes, we have constructed plasmids that allow us to define the DNA signals required for initiation and termination of VA, RNA transcription. We have found an internal control region for VA, transcription initiation located close to the 5' end of the respective gene, as determined by an ordered set of deletions constructed by using the nuclease BAL 31.MATERIALS AND METHODS DNA Templates. Restriction enzyme fragments were prepared from whole adenovirus 2 (Ad2) DNA purified on CsCl density gradients as described (16) and inserted into plasmid pBR322. We constructed a wild-type clone containing the Ad2 sequences from the Sal I site (coordinate 26.9) to the HindIII site (coordinate 31.5) on the physical Ad2 map which goes from 0 to 100. dl 5' +70 (deletion of all sequences upstream from nucleotide +70) was ...

Human adenovirus early region 1A (E1A) gene products differentially regulate the expression of early region 2A (E2A) encoding the DNA‐binding protein (DBP). In a microinjection system, plasmids containing the DBP gene associated with both its early (map coordinate 75) and late (coordinate 72) promoters, or only with the early promoter, are inefficiently expressed, and the presence of E1A DNA is required for full expression. In contrast, the E2A plasmid in which the DBP gene is associated solely with its late promoter, efficiently produces DBP, the synthesis of which is significantly inhibited by an E1A gene product. To identify which of the E1A products is responsible for either activation or repression of DBP gene expression, two E1A mutants (Ad5hr1 and Ad2/5pm975) have been tested in the microinjection system in the presence of different DBP plasmids containing either one or both promoters. The results obtained indicate that the product encoded by the E1A 13S mRNA is responsible for the stimulation of DBP produced from the early promoter and that the 12S mRNA codes for the product which represses the synthesis of DBP from the late promoter. These results were confirmed using clones in which the E2A early or late promoter was associated to the chloramphenicol acetyltransferase (CAT) gene and assayed for CAT activity after cell transfection in the absence or in the presence of wild‐type or mutant E1A plasmids, and we have also shown that this promoter‐dependent regulation is reflected in the relative amount of specific DBP mRNA.

A method is described which permits the ligation- mediated PCR amplification of specific fragments from a Class-II restriction endonuclease total digest. Feasibility was tested using Bcl I and phage lambda DNA as a model enzyme and amplicon system, respectively. Bcl I is one of many widely used restriction enzymes which cleave at palindromic recognition sequences and leave 5'-protruding ends of defined sequence. Using a single pair of universal primers, a given fragment can be specifically amplified after joining the fragments to adaptors consisting of a duplex primer region and a 9-nucleotide protruding single-stranded 5'-end containing the sequence complementary to the cleaved restriction site and a 4-nucleotide 'indexing sequence.' The protruding strand anneals to a restriction fragment by displacing its corresponding strand in the same fragment-specific indexing sequence located juxtaposed to the restriction site. The adaptor is covalently linked to the restriction fragment by T4 DNA ligase, and amplification is carried out under conditions for long-distance PCR using the M13 forward and reverse primers. The technique discriminated robustly between mismatches and perfect matches for the 16 indexing sequences tested to allow individual lambda Bcl I fragments to be amplified from their respective adaptor pairs. A strategy is proposed enabling a non-cloning approach to the accession, physical mapping and sequencing of genomic DNA. The method could also have application in high-throughput genetic mapping and fingerprinting and should expand the enzyme base for ligation- mediated indexing technology which has previously been limited to the Class-IIS and IP restriction endonucleases.