City of Hope

R Bruce Wallace

John J Rossi

John A Zaia

Luis A Ugozzoli

Edouard M Cantin

Bijay K Pal

George J Murakawa

Dan Y Wu

A process for identifying a viral RNA nucleotide sequence present in a sample of peripheral blood cells which comprises amplifying such RNA simultaneously with at least one other RNA nucleotide sequence present in a virus infected cell in said sample, and thereafter separately and sequentially analyzing the amplification reaction products with probes homologous with authentic RNA and with such other RNA sequence to identify one or both of said RNA nucleotide sequences.

Method for amplification and detection of RNA sequences

A method is described for the identification of point mutations by a template-dependent ligation procedure. Also described is a template-dependent ligase chain reaction procedure for the amplification and detection of nucleic acid sequences.

Ligation amplification of nucleic acid sequences

The present invention is directed to a method for detecting the presence or absence of any specific target nucleic acid sequence contained in a sample. The target sequence can be present in the sample in a relatively pure form or as a component of a member of a mixture of different nucleic acids. The method of the invention utilizes a novel primer design. The sequence of the novel primer is composed of two portions, the 3&prime; portion is a primer specific for the desired nucleic acid sequence and the 5&prime; portion is complementary to preselected nucleic acid sequence. Extension of the 3&prime; portion of the primer with a labeled deoxynucleosides triphosphate yields a labeled extension product if, but only if, the template includes the target sequence. The labeled extension product is detected by hybridization of the 5&prime; portion to the preselected sequence. The preselected sequence is preferably bound to a solid support as one member of a grid having a group of sequences.

Method of detecting and discriminating between nucleic acid sequences

Discrimination between RNAs which differ by as little as a single nucleotide is accomplished by having an excess of unlabeled non-complementary oligonucleotide present during hybridization with a labeled complementary nucleotide. The non-complementary oligonucleotide blocks hybridization of the labeled complementary oligonucleotide sequence without affecting hybridization of the labeled complementary oligonucleotide to the complementary sequence. The label may be an isotope, a fluorescent group or of any other type. The technique permits examination of the transcription of highly related allelic and non-allelic genes present in the same cell and the quantification of such transcripts by use of appropriate internal control. The technique is also useful in oligonucleotide hybridization to DNA sequences which differ only by a single nucleotide.

Competitive hybridization technique

A rapid, non-radioactive approach to the diagnosis of sickle cell anemia is described based on an allele specific polymerase chain reaction (ASPCR) in which the 3'-terminal nucleotide of one of the primers of the primer set forms a match with one allele and a mismatch with the other allele. This method allows the direct detection of the normal or the sickle cell .beta.-globin allele in genomic DNA without the additional steps of probe hybridization, ligation or restriction enzyme cleavage.

Allele specific polymerase chain reaction

Disclosed is an application of allele specific polymerase chain reaction technology for the direct determination of multiple allele genotyping. ABO genotyping is demonstrated with allele specific primer sets.

Genotyping of multiple allele systems

A method of inhibiting human immunodeficiency virus (HIV) comprising administering a therapeutically effective amount of an antiviral agent to attack the first splice acceptor site of the tat III gene of HIV.

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Greenbrae, CA, United States of America

R Bruce Wallace