Single-base mutational analysis of cancer and genetic diseases using membrane bound modified oligonucleotides.

A convenient format for the detection of PCR amplified sequences is the hybridization of the PCR products to oligonucleotide probes which are immobilized on a solid phase. We describe a new method for site-specific attachment of such probe oligonucleotides to nylon membranes. The method is based on the formation of an amide bond between carboxyl groups present on the membranes and amino-linkers situated on the 5' end of the oligonucleotides. The covalent attachment is via a carbodiimide mediated condensation. The single, 5' end attachment of the oligonucleotides to the membrane surface leaves the probe free to interact with complementary sequences, thus increasing the hybridization efficiency relative to methods where heat or ultraviolet light is used for non-specific fixation. Using biotinylated PCR products in hybridization reactions along with a non-radioactive chemiluminescent detection system, high efficiency hybridization is obtained as well as a very good signal to noise ratio. The method has been applied successfully to the detection of RAS point mutations, cystic fibrosis deletion and point mutations and others. The sensitivity, simplicity and reproducibility of this method make it an ideal tool for the diagnosis of infectious and genetic diseases, as well as analysis of mutations in neoplasias, HLA typing and other areas.

Genetic analysis of amplified DNA with immobilized sequence-specific oligonucleotide probes.

The analysis of DNA for the presence of particular mutations or polymorphisms can be readily accomplished by differential hybridization with sequence-specific oligonucleotide probes. The in vitro DNA amplification technique, the polymerase chain reaction (PCR), has facilitated the use of these probes by greatly increasing the number of copies of target DNA in the sample prior to hybridization. In a conventional assay with immobilized PCR product and labeled oligonucleotide probes, each probe requires a separate hybridization. Here we describe a method by which one can simultaneously screen a sample for all known allelic variants at an amplified locus. In this format, the oligonucleotides are given homopolymer tails with terminal deoxyribonucleotidyltransferase, spotted onto a nylon membrane, and covalently bound by UV irradiation. Due to their long length, the tails are preferentially bound to the nylon, leaving the oligonucleotide probe free to hybridize. The target segment of the DNA sample to be tested is PCR-amplified with biotinylated primers and then hybridized to the membrane containing the immobilized oligonucleotides under stringent conditions. Hybridization is detected nonradioactively by binding of streptavidin-horseradish peroxidase to the biotinylated DNA, followed by a simple colorimetric reaction. This technique has been applied to HLA-DQA genotyping (six types) and to the detection of Mediterranean beta-thalassemia mutations (nine alleles).

Diagnosis of sickle cell anemia and beta-thalassemia with enzymatically amplified DNA and nonradioactive allele-specific oligonucleotide probes.

We have developed a simple and rapid nonradioactive method for detecting genetic variation and have applied it to the diagnosis of sickle cell anemia and beta-thalassemia. The procedure involves the selective amplification of a segment of the human beta-globin gene with oligonucleotide primers and a thermostable DNA polymerase, followed by hybridization of the amplified DNA with allele-specific oligonucleotide probes covalently labeled with horseradish peroxidase. The hybridized probes were detected with a simple colorimetric assay. We demonstrated the usefulness of this method in a retrospective analysis of two pregnancies at risk for beta-thalassemia and one at risk for sickle cell anemia, as well as in an analysis of nine DNA samples simulating three family sets.

Nucleosides of 1,4-thiazin-3-one and derivatives as tetrahedral intermediate analogues of enzymes in pyrimidine nucleoside metabolism.

Reaction of the trimethylsilylated derivative of 1,4-thiazin-3-one with 1-O-acetyl-2,3,5-tri-O-benzoyl-D-ribofuranose in the presence of SnCl4 gave, after deblocking, 4-beta-ribofuranosyl-1,4-thiazin-3-one (8). Treatment of 1,4-thiazin-3-one with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose in the presence of sodium hydride provided, after deblocking, the corresponding 2-deoxy-beta-D-ribofuranosyl derivatives (19). Oxidation of 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazin-3-one (7) with 1 equiv of m-chloroperbenzoic acid resulted in 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazine-2,3-dione (9) and 4-(2,3,5-tri-O-benzoyl-beta-D-ribofuranosyl)-1,4-thiazin-3-one 1-oxide (10). Evidence is presented that indicates that the oxidation of the thiazine at the 2-position is due to a Pummerere rearrangement. The new compounds failed to show significant activity against tumor cell lines in culture, L1210 cells in vivo, virus cytotoxicity in cell culture, or cytidine deaminase.

Use of nonisotopic M13 probes for genetic analysis: application to HLA class II loci.

Previously, DNA polymorphisms in the HLA gene cluster have been analyzed using radioactive probes in Southern blot experiments; the restriction fragment length polymorphisms (RFLPs) revealed by this analysis are capable of subdividing HLA serological types. Here, we report the use of DNA probes labeled with biotinylated psoralen to provide nonisotopic detection of HLA class II RFLP patterns. These biotinylated probes contain cDNA sequences encoding the alpha and beta chains of DP, DQ, and DR HLA class II genes as inserts in M13 vectors. The recombinant M13 molecules are partially double-stranded with single-stranded HLA cDNA regions and contain biotinylated psoralen covalently linked to duplex DNA by UV irradiation. Following hybridization, the presence of biotinylated probe bound to target DNA is detected using a streptavidin-horseradish peroxidase conjugate, which converts the colorless substrate 3,3',5,5'-tetramethylbenzidine to a blue precipitate in less than 1 hr. The probe and detection system described here can detect single-copy genes in less than 0.5 microgram of total human DNA on Southern blots and generates the same specific RFLP patterns as do probes labeled with 32P by nick-translation. These biotinylated HLA class II probes have been applied to tissue typing for bone marrow transplantation and the study of insulin-dependent diabetes susceptibility, revealing in each case relevant polymorphisms not detected by serologic typing.

Design and synthesis of tetrahedral intermediate analogues as potential dihydroorotase inhibitors.

Three new heterocyclic analogues (4-6) of dihydroorotic acid were designed, synthesized, and tested as inhibitors of dihydroorotase. Each compound possessed a tetrahedral sulfur atom at the position equivalent to carbon 4 in the dihydroorotate ring in an attempt to mimic the presumed tetrahedral transition state in the course of the enzymatic reaction. Additionally, N-carbamyl-3-phosphonoalanine was prepared and evaluated as a dihydroorotase inhibitor. Compounds 4 and 6 were modest inhibitors (I50's of 0.52 and 0.18 mM, respectively), but the other candidate inhibitors showed little inhibition at 1 mM.