Large-scale SNP scoring from unamplified genomic DNA.

Discoveries from the Human Genome Project (HGP) continue to spur changes in medical technology that will lead to new diagnostic procedures in the clinical lab. As more single nucleotide polymorphisms (SNPs) are discovered and correlated to human diseases, demands for genetic tests will increase. The enormity of the number of SNPs makes developing inexpensive and reliable high-throughput methods for SNP scoring imperative. High-throughput screening (HTS) means, at a minimum, a production rate of thousands of assays per day. Ideally, the technology will be easy, inexpensive and amenable to automation. The Invader assay offers a simple diagnostic platform to detect single nucleotide changes with high specificity and sensitivity from unamplified, genomic DNA. The Invader assay uses a structure-specific 5' nuclease (or flap endonuclease) to cleave sequence-specific structures in each of two cascading reactions. The cleavage structure forms when two synthetic oligonucleotide probes hybridise in tandem to a target. One of the probes cycles on and off the target and is cut by the nuclease only when the appropriate structure forms. These cleaved probes then participate in a second Invader reaction involving a dye-labelled fluorescence resonance energy transfer (FRET) probe. Cleavage of this FRET probe generates a signal, which can be readily analysed by fluorescence microtitre plate readers. The two cascading reactions amplify the signal significantly; each original target molecule can lead to more than 10(6) cleaved signal probes in one hour. This signal amplification permits identification of single base changes directly from genomic DNA without prior target amplification. The sequences of the oligonucleotide components of the secondary reaction are independent of the target of interest and permit the development of universal secondary reaction components useful to identify any target.

The zygotic control of Drosophila pair-rule gene expression. II. Spatial repression by gap and pair-rule gene products.

We examined gene expression patterns in certain single and double pair-rule mutant embryos to determine which of the largely repressive pair-rule gene interactions are most likely to be direct and which interactions are probably indirect. From these studies we conclude that: (i) hairy+ and even-skipped (eve+) regulate the fushi tarazu (ftz) gene; (ii) eve+ and runt+ regulate the hairy gene; (iii) runt+ regulates the eve gene; but, (iv) runt does not regulate the ftz gene pattern, and hairy does not regulate the eve gene pattern. These pair-rule interactions are not sufficient, however, to explain the periodicity of the hairy and eve patterns, so we examined specific gap gene mutant combinations to uncover their regulatory effects on these two genes. Our surprising observation is that the hairy and eve genes are expressed in embryos where the three key gap genes hunchback (hb), Krüppel (Kr), and knirps (kni) have been removed, indicating that these gap genes are not essential to activate the pair-rule genes. In fact, we show that in the absence of either hb+ or kni+, or both gap genes, the Kr+ product represses hairy expression. These results suggest that gap genes repress hairy expression in the interstripe regions, rather than activate hairy expression in the stripes. The molecular basis of pair-rule gene regulation by gap genes must involve some dual control mechanisms such that combinations of gap genes affect pair-rule transcription in a different manner than a single gap gene.

The zygotic control of Drosophila pair-rule gene expression. I. A search for new pair-rule regulatory loci.

The examination of pair-rule gene expression in wild-type and segmentation mutant embryos has identified many, but not necessarily all, of the elements of the regulatory system that establish their periodic patterns. Here we have conducted a new type of search for previously unknown regulators of these genes by examining pair-rule gene expression in blastoderm embryos lacking parts of or entire chromosomes. This method has the advantage of direct inspection of abnormal pair-rule gene patterns without relying upon mutagenesis or interpretation of larval phenotypes for the identification of segmentation genes. From these experiments we conclude that: (i) most zygotically required regulators of the fushi tarazu (ftz), even-skipped (eve) and hairy (h) pair-rule genes have been identified, except for one or more loci we have uncovered on chromosome arm 2L; (ii) the repression of the ftz and eve genes in the anterior third of the embryo is under maternal, not zygotic control; and (iii) there are no general zygotically required activators of pair-rule gene expression. The results suggest that the molecular basis of pair-rule gene regulation can be pursued with greater confidence now that most key trans-acting factors are already in hand.