Abstract: Methods are provided for enhanced specificity and sensitivity of nucleic acid amplification. The methods are simplified nested amplification procedures wherein both inner and outer primer pairs are present in the amplification reaction mixture. According to the methods, the thermocycling profile, as well as the sequences, length, and concentration of amplification primers, is modified to regulate which primers are annealed and extended on the target during any particular amplification cycle. The methods described are particularly suitable in PCR amplifications and have numerous applications in molecular biology, medical diagnostics, and forensics.

Abstract: Liquid perfluorocarbon supports useful as liquid affinity supports are provided. The support is based on an inert perfluorocarbon carrier with ligands or binders for the ligands attached to its surface through a highly fluorinated isocyanate anchor group.

Abstract: A method and a kit for the isolation and quantitative detection of a selected target nucleic acid sequence from solution employing two probes. A first probe is complementary to one portion of the target and is covalently attached to a first complexing agent (e.g., either an antigen or an antibody). The second probe is complementary to a different portion of the target and is associated with a reporter group. Following hybridization of the target and two probes in solution, a solid support coated with a second complexing agent (i.e., a corresponding antibody or antigen) capable of binding to the first complexing agent on the first probe is employed to immobilize the target-probe hybrid complex. A plurality of types of first probes may be used. Each type is attached to the same sort of complexing agent but each includes a nucleic acid sequence which is complementary to a different portion of the target.

Abstract: An autoradiographic gene-screening method employing a hybridization process, which comprises:(1) a step of transferring at least a portion of nucleic acids, fragments thereof or derivatives thereof resolved on a medium onto a transfer support to fix them thereonto;(2) a step of hybridizing the nucleic acids, fragments thereof or derivatives thereof fixed onto said transfer support with radioactively labeled probes; and(3) a step of obtaining locational information on the radioactively labeled substances on said transfer support, which comprises placing said transfer support having been subjected to the hybridization and a stimulable phosphor sheet in layers for a given period of time to cause said sheet to absorb at least a portion of radiation energy emitted by the radioactively labeled substances on said transfer support, exciting said stimulable phosphor sheet with an electromagnetic wave to release the radiation energy stored in said sheet as stimulated emission, and detecting the stimulated emission.

Abstract: A nucleic acid construct useful in preparing reagents for determining target nucleotide sequences in the nucleic acid of a biological sample, the construct having in its single-stranded form:(a) a target binding region substantially complementary to the target nucleotide sequence, and(b) a signal strand pairing segment bound in the construct by complementary base pairing to a portion of the target binding region;a second portion of the target binding region being single-stranded; andthe target binding region and signal strand pairing segment being covalently linked by a phosphate/sugar backbone.A replicable nucleic acid having an origin of replication and two half-restriction sites capable of forming a restriction site can be treated with a restriction enzyme to form a length of nucleic acid containing the target binding region and the signal strand pairing segment. Subsequent labeling of the construct and various optional cleavage and derivation steps can convert the construct to a reagent complex.

Abstract: Methods for preparing liquid supports utilized in bioaffinity and ion-exchange separation methods and provided. The support is based on an inert carrier with ligands or binders for ligands attached through its surface through a highly fluorinated isocyanate anchor group. The use of such supports in capturing neutral and charged target molecules from samples and in analytical applications are also provided.

Abstract: Bioaffinity and ion-exchange separation methods are provided along with liquid supports utilized in these methods. The support is based on an inert carrier with ligands or binders attached to its surface through a highly fluorinated isocyanate anchor group. Methods for preparing such supports and their use in capturing neutral and charged target molecules from samples and in analytical applications are also provided.

Abstract: An assay for polynucleotides employing total internal reflection of excitation radiation at a coating bonded to the surface of an optically conductive glass cell. The coating initially includes single-stranded polynucleotides coupled to individual attachment sites on the surface of the cell, such polynucleotides being complementary, at least in part to the single-stranded form of the polynucleotide that is being assayed. Each molecule of the coupled polynucleotide is connected to the cell surface through a spacer connected to an irreversibly conjugated polyadenine/polythymidine sequence at one end of the coupled polynucleotide.When the surface of the coated cell is contacted with a sample that contains single-stranded polynucleotide complementary to the bound polynucleotide, renaturation will occur, forming a double-stranded form of the polynucleotide of interest.

Abstract: Disorders of the base sequences in genomic substances such as DNA and RNA are detected by changing the state of aggregation of fine particles by cleavaging using a nuclease. A single-stranded denatured product of the objective genomic substance is added to first and second fine particles each attached to plural pieces of first and second single-stranded nucleic acid probes, respectively. The first and second single-stranded nucleic acid probes are complementary to a first region and a second region, respectively, on the objective genomic substance, which are exclusive of each other and contiguous from each other. Aggregations of the first and second fine particles are formed by double or multiple hybridization reaction of the denatured objective genomic substance added with the first and second single-stranded nucleic acid probes.

Abstract: The cDNA coding for PP15 is described. This cDNA can be used to prepare PP15 in pro- or eukaryotic cells.

Abstract: A method for making synthetic oligonucleotides which bind to target sequences in a duplex DNA forming colinear triplexes by binding to the major groove. The method includes scanning genomic duplex DNA and identifying nucleotide target sequences of greater than about 20 nucleotides having either about at least 65% purine bases or about at least 65% pyrimidine bases; and synthesizing synthetic oligonucleotides complementary to identified target sequences. The synthetic oligonucleotides have a G when the complementary location in the DNA duplex has a GC base pair and have a T when the complementary location in the DNA duplex has an AT base pair. The synthetic oligonucleotides are oriented 5' to 4' and bind parallel or 3' to 5' and bind anti-parallel to the about at least 65% purine strand.

Abstract: The presence or absence of a nucleic acid sequence associated with one or more related viruses in a sample containing one or more nucleic acids and suspected of containing such sequence can be detected by amplifying the sequence using primers to form extension products as templates and detecting the amplified product if it is present. This may be accomplished by adding a labeled hybridization probe to the amplified product either free in solution or after immobilization on a solid support. Preferably the virus constitutes AIDs viruses and hepadnaviruses.

Abstract: A method for the formation of double stranded nucleic acid molecules from separate single stranded nucleic acid molecules in a single phase reaction solution is disclosed wherein the rate of reaction is greatly increased over the rate of reaction at standard reference conditions. The greatly accelerated reaction rate is accomplished through the use of known concentrations of nucleic acid precipitating agents which are added to the reaction solution. Nucleic acid denaturing agents may also be added. The solution so formed is incubated and then assayed for the presence of double stranded nucleic acid molecules.

Abstract: This invention relates to an improved process for amplifying a specific nucleic acid sequence. The process involves synthesizing single-stranded RNA, single-stranded DNA and Double-stranded DNA. The single-stranded RNA is a first template for a first primer, the single-stranded DNA is a second template for a second primer, and the double stranded DNA is a third template for synthesis of a plurality of copies of the first template. A sequence of the first primer or the second primer is complementary to a sequence of the specific nucleic acid and a sequence of the first primer or the second primer is homologous to a sequence of the specific nucleic acid. The improvement of the amplification process involves the addition of DMSO alone or in combination with BSA, which improves the specificity and efficiency of the amplification.

Abstract: A single-stranded nucleic acid molecule which encodes an amino acid sequence comprising at least a portion of a T4 glycoprotein is provided. Additionally, amino acid sequences which comprise at least a portion of a T4 glycoprotein and are useful as a prophylaxis for treating a subject with acquired immune deficiency syndrome are provided. These amino acid sequences, are capable of specifically forming a complex with a human immunodeficiency virus envelope glycoprotein and which are soluble in an aqueous solution. Monoclonal antibodies directed to the water-soluble amino acid sequences of the present invention may be used as vaccines for immunizing a subject against acquired immune deficiency syndrome.

Abstract: A probe capable of binding by homologous base pairing independently to a pair of gene regions bordering the upstream and downstream sides of a pair of infrequent restriction endonuclease sites separated by between about 20-2,000 kilibases on a section of linear DNA. The probe is produced, according to the method of the invention by digesting the DNA section to completion with the selected endonuclease, and ligating the resulting fragments under conditions which favor end-to-end circularization, and selecting digest fragments of the large circular molecules which have end to end junctions. Also disclosed are method for mapping and ordering the positions of such probes, using another set of linking probes which span such rate cutting sites.

Abstract: DQ beta gene oligonucleotides consisting essentially of the sequence GGCCGCCTGCCGCCGAG or of the sequence GCTGGGGCTGCCTGCCG and the use of such oligonucleotides in a method for assaying for a polymorphic region associated with Type I diabetes mellitus in humans.

Abstract: Repetitive DNA sequences of the protozoan parasites T. cruzi and P. falciparum have been isolated. These sequences appear more than once in the parasitic organism. Once isolated, the DNA sequences may be labelled with materials such as chromogens, biotin, or radioactive substances. The sequences may be used, in labelled or unlabelled form, as probes for the parasites.

Abstract: A method for sequencing nucleic acid polymers is provided in which the polymer to be sequenced acts as a template for the production of a complementary polymer by a polymerase enzyme. The template polymer is introduced into a polymerization environment in which production of the complementary polymer will occur if appropriate nucleotides are provided. The nucleotides are then provided to the polymerization environment one at a time in individual feedstocks. If the nucleotide in a feedstock is complementary to the next base in the template polymer, i.e., the unpaired base closest to the growing end of the complementary polymer, polymerization will occur lengthening the complementary polymer and releasing PPi. By separately recovering each feedstock and analyzing it for the presence of PPi, the sequence of the complementary polymer and thus the template polymer is determined.

Abstract: DQ beta gene oligonucleotides selected from the group consisting of(1) GAGAGGAGTACGCACGCTT,(2) GCTGGGGCCGCCTGCCGCC,(3) AGGACCCGGGCGGAGTTGG,(4) GAGAAGAGATCGTGCGCTT,(5) GCTGGGGCTGCCTGCCGCC and(6) AGGAAACGGGCGGCGGTGG.Such oligonucleotides are useful in methods for detecting the proclivity in a human for development of Type I diabetes mellitus.