Showing papers on "Exome sequencing published in 1998"

Rapid sequencing of the p53 gene with a new automated DNA sequencer

Abstract: p53 is the most commonly mutated gene in human cancers. Approximately 90% of the p53 gene mutations are localized between domains encoding exons 5 to 8. Sequencing methods currently available are tedious and time-consuming and are not suitable for routine laboratory testing. In an effort to identify a simple and rapid sequencing method, we analyzed 16 preselected breast tumors and 18 preselected ovarian tumors, using a newly developed automated DNA sequencer. p53 gene mutations had been previously identified in these tumors, using a conventional automated sequencing procedure. Exons 5 to 8 were amplified by PCR, and the PCR products were subsequently subjected to cycle sequencing with the Sanger chain termination method, using Cy5.5-labeled primers. The sequencing mixture was then resolved on a newly developed automated DNA sequencer that can sequence ∼300 bases of DNA in 30 min. Of these 16 breast tumors, two had mutations in exon 5, four in exon 6, three in exon 7, and three in exon 8. Of the 18 ovarian tumors, two had mutations in exon 5, five in exon 6, two in exon 7, and three in exon 8. In all cases, we identified the same mutations by both the new and the conventional sequencing procedures. Most mutations affected an arginine codon. These data demonstrate that the new method has the capability to provide accurate sequencing information in a fraction of the time and labor in comparison with current automated sequencing techniques. When such procedures are used, DNA sequencing may become a routine tool for identifying clinically important mutations for diagnosis and prognosis of patients with genetic, malignant, infectious, and other diseases.

Method and kit for amplification, sequencing and typing of classical hla class i genes

Abstract: Amplification primer and sequencing primer sites have been identified which permit sequence-based typing of each of the classical HLA genes in highly robust and consistent reactions without allelic dropout. To determine the DNA sequence, and thus the type of at least one exon of an HLA-A, HLA-B and HLA-C gene present in a sample, the sample is combined with an amplification reaction mixture containing the amplification primers and amplified to form an amplification product including exon 2 and exon 3 of the gene together in a single fragment. The amplification product is then combined with a sequencing reaction mixture containing one or more oligonucleotide sequencing primers which hybridize to conserved regions of the amplification product. The oligonucleotide sequencing primers between them are effective to produce sequencing fragments from all known alleles of exon 2 or exon 3 of the gene under suitable conditions to produce sequencing fragments. The sequencing fragments are then evaluated to determine the sequence of exon 2 or exon 3 of the gene in the sample.

Doublex Sequencing in Molecular Diagnosis of Hereditary Diseases

Abstract: We describe doublex sequencing of human genomic PCR products using two differently labeled primers in a single reaction and analysis on two automated DNA sequencing devices. Feasibility of the methodology is demonstrated by isothermal and cycle sequencing for two different PCR products and by cycle sequencing on both strands of a single product. It was applied to analyze mutations in patient DNAs in routine sample screening. Because it has the advantage of increased throughput and cost reduction while retaining its accuracy and reading length, we found that doublex sequencing is an attractive option for molecular diagnosis of hereditary diseases. This approach would be even more beneficial if it used DNA sequencing devices with several lasers in a single instrument.

Cloning and sequencing of human CD14 gene

Abstract: Human CD14 gene was cloned from genomic DNA of PMAstimulated HL60 cells by PCR. Sequencing result showed that it is identical to the reported human CD14 gene except four bases, among which two new mutations were identified.