X-linked Alport syndrome is a hereditary glomerulonephritis in which progressive loss of kidney function is often accompanied by progressive loss of hearing. Ultrastructural defects in glomerular ...basement membranes (GBM) of Alport syndrome patients implicate an altered structural protein as the cause of nephritis. The product of COL4A5, the α5(IV) collagen chain, is a specific component of GBM within the kidney, and the gene maps to the same X chromosomal region as does Alport syndrome. Three structural aberrations were found in COL4A5, an intragenic deletion, a Pst I site variant, and an uncharacterized abnormality, which appear to cause nephritis and deafness, with allele-specific severity, in three Alport syndrome kindreds in Utah.
Methods are described for cloning random or highly degenerate nucleotide (nt) sequences. The procedures use synthetically derived mixtures of oligodeoxynucleotides (oligos) whose heterogeneous ...central portions are bounded at their 5' and 3' ends by sequences recognized by restriction endonucleases. Oligo collections of defined length and nt composition are synthesized by utilizing appropriate concentrations of all four nucleotide precursors during each addition step for the central region. Single-stranded oligos with appropriate 5' and 3' ends can be ligated directly, although inefficiently, into double-stranded (ds) DNA molecules with complementary 5' and 3' extensions produced by restriction endonuclease cleavage. A more general and efficient method is to convert the oligo into a ds form by incubating it with the Klenow (large) fragment of Escherichia coli DNA polymerase I. If the 3' ends are palindromic, two oligo molecules will serve as mutual primers for polymerization. The resulting products are ds molecules containing two oligo units separated by the original 3' restriction site and bounded at each end by the original 5' restriction site. After appropriate restriction endonuclease cleavage, oligo units can be cloned by standard procedures. Analysis of 26 recombinant M13 phages indicates that the nt sequences of the cloned oligos are in good accord with what was expected on a random basis.
Random-sequence or highly degenerate oligonucleotides have been useful for defining functionally important sequences both in proteins and in nucleic acids. In this approach, such oligonucleotides are ...used to replace a segment of DNA required for a desired function, and functional sequences are identified by an appropriate genetic or biochemical selection. Here, a collection of 105 altered β -lactamase proteins was generated by cloning a mixed-base oligonucleotide in place of the sequences coding for a 17-amino acid portion of the enzyme's active site. Approximately 2000 enzymes from this collection were able to confer ampicillin resistance on Escherichia coli. Fifty-eight of these were chosen for further study after characterization with various β -lactam substrates. β -Lactamases having altered specificity against different antibiotics, resistance to the suicide inhibitors clavulanic acid and sulbactam, and temperature-dependent activities were obtained. The amino acid residues responsible for these altered properties as well as for basic enzyme activity are defined. This approach should prove to be an effective and general tool for creating proteins with novel properties, especially in situations in which a high-resolution structure of the protein is not known.
The Human Genome Project has opened the door to personalized medicine, provided that human genetic diversity can be analyzed in a high-throughput and cost-effective way Illumina has developed a ...genotyping system that combines very high throughput and accuracy with low cost per SNP analysis. The system uses our BeadArray platform, a high level of multiplexing, and modular, scalable automation to meet the requirements for cost-effective, genome-wide linkage disequilibrium studies. As implemented in a high-throughput genotyping service facility at Illumina, the system has a current capacity of one million SNP assays per day and is easily expandable. Each SNP call is associated with a quality score that correlates with accuracy
We have developed a flexible, accurate and highly multiplexed SNP genotyping assay for high-throughput genetic analysis of large populations on a bead array platform. The novel genotyping system ...combines high assay conversion rate and data quality with >1500 multiplexing, and Array of Arrays formats. Genotyping assay oligos corresponding to specific SNP sequences are each linked to a unique sequence (address) that can hybridize to its complementary strand on universal arrays. The arrays are made of beads located in microwells of optical fiber bundles (Sentrix Array Matrix) or silicon slides (Sentrix BeadChip). The optical fiber bundles are further organized into a matrix that matches a 96-well microtiter plate. The arrays on the silicon slides are multi-channel pipette compatible for loading multiple samples onto a single silicon slide. These formats allow many samples to be processed in parallel. This genotyping system enables investigators to generate approximately 300,000 genotypes per day with minimal equipment requirements and greater than 1.6 million genotypes per day in a robotics-assisted process. With a streamlined and comprehensive assay, this system brings a new level of flexibility, throughput, and affordability to genetic research.
Decoding randomly ordered DNA arrays Gunderson, Kevin L; Kruglyak, Semyon; Graige, Michael S ...
Genome research,
05/2004, Letnik:
14, Številka:
5
Journal Article
Recenzirano
Odprti dostop
We have developed a simple and efficient algorithm to identify each member of a large collection of DNA-linked objects through the use of hybridization, and have applied it to the manufacture of ...randomly assembled arrays of beads in wells. Once the algorithm has been used to determine the identity of each bead, the microarray can be used in a wide variety of applications, including single nucleotide polymorphism genotyping and gene expression profiling. The algorithm requires only a few labels and several sequential hybridizations to identify thousands of different DNA sequences with great accuracy. We have decoded tens of thousands of arrays, each with 1520 sequences represented at approximately 30-fold redundancy by up to approximately 50,000 beads, with a median error rate of <1 x 10(-4) per bead. The approach makes use of error checking codes and provides, for the first time, a direct functional quality control of every element of each array that is manufactured. The algorithm can be applied to any spatially fixed collection of objects or molecules that are associated with specific DNA sequences.
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