Orchid Biosciences, Inc.

University of Minnesota

Beckman Coulter, Inc.

Orchid Biocomputer, Inc.

Michael T Boyce-Jacino

Pedro Santamaria

Jose J Barbosa

Stephen S Rich

Anthony J Faras

Philip Goelet

Steven R Head

Jufang Shi

Jonathan Karn

Yu-Hui Rogers

Mitchell A Friedman

Phillip Goelet

Miriam B Addelston

The present invention describes a novel nucleic acid sequencing reagent which consists of a capture moiety, a spacer region and a sequence specific hybridizing region of 4-8 bases. The nucleic acid sequencing reagent of the present invention can also contain an attachment moiety. The nucleic acid sequencing reagent can be arranged into a nested array. This array configuration can then be used to sequence a given template without prior knowledge (de novo) of the wild type or expected sequence in conjunction with primer extension in the presence of a labeled chain terminating nucleotide.

De novo or 'universal' sequencing array

The present invention relates to the field of nucleic acid sequence analysis. The invention provides methods for performing a polymerase signal assay (PSA) to analyze nucleotide sequences using solid phase sequence arrays comprising a plurality of sequence reagents with primer sequences 4-6 bases in length. The methods of the invention generate a binary signal pattern which can be used to identify nucleic acid sequences and/or mutations and polymorphisms of a nucleic acid sequence. Mutations and polymorphisms which can be identified by the methods of the invention include single nucleotide polymorphisms (SNP's), base deletions, base insertions, and heterozygous as well homozygous polymorphisms.

Polymerase signaling assay

The invention relates to a simple, cost effective method for immobilizing synthetic nucleic acid molecules onto a solid support The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.

Covalent attachment of unmodified nucleic acids to silanized solid phase surfaces

De novo or &ldquo;universal&rdquo; sequencing array

The present invention provides a method for determining the nucleotide sequence of a nucleic acid molecule using primer oligonucleotides arrayed on a solid support and doing primer extension. By providing sequence information from both oligonucleotide primers in which the 3&prime; terminal nucleotide of the oligonucleotide primer is hybridized to the nucleic acid molecule and oligonucleotide primers in which the 3&prime; terminal nucleotide is not hybridized to the nucleic acid molecule the invention is advantageous in that less primers are needed to fully sequence a nucleic acid molecule. Where the 3&prime; terminal nucleotide of an oligonucleotide in the array does not hybridize to the nucleic acid molecule, the present invention provides for either not permitting oligonucleotides hybridized to nucleic acid molecules to be extended by polymerase-mediated incorporation of a single chain terminator nucleotide residue onto the 3&prime; terminus of said hybridized oligonucleotide, or permitting the removal of any non-hybridized nucleotide residues from the 3&prime; terminus of said hybridized oligonucloetides, so as to form a truncated primer oligonucleotide whose 3&prime; terminus is hybridized to said nucleic acid molecule. The invention also provides kits that can be used to implement the methods of the invention.

Method for analyzing the nucleotide sequence of a polynucleotide by oligonucleotide extension on an array

The invention concerns a novel class of 3&prime;-modified, pro-fluorescent nucleotides. The invention also pertains to methods for using such nucleotides.

Nucleotide analogues with 3'-pro-fluorescent fluorophores in nucleic acid sequence analysis

The invention concerns an apparatus for printing arrays of molecules and methods for printing these arrays. More particularly, the invention relates to a gene pen apparatus that may be used for delivering arrays of oligonucleotides on solid supports from a multiplicity of reagent reservoirs through flow controlling means. The flow controlling means may for example, be either a pin valve means or a wicking means.

Gene pen devices for array printing

This invention relates to a composition having a nucleic acid molecule covalently coupled to a solid support by means of a covalent linkage. The nucleic acid molecule is covalently coupled to the solid support by a silane. The composition can be used in nucleic acid hybridization assays and sequencing by hybridization assays.

Covalent attachment of unmodified nucleic acids to silanized solid phase

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of CDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed.

DNA sequence-based HLA typing method

The invention relates to a simple, cost effective method for immobilizing synthetic nucleic acid molecules onto a solid support. The invention further concerns the use of such immobilized molecules in nucleic acid hybridization assays, sequencing by hybridization assays, and genetic analyses and combinatorial analyses involving nucleic acids or proteins for screening applications.

Attachment of unmodified nucleic acids to silanized solid phase surfaces

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class II and Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of DRB, DQB, DQA, DPB, DPA, HLA-A, HLA-B and HLA-C- transcripts enzymatically amplified using a limited number of non-allele-specific oligonucleotides. Total cellular RNA from peripheral blood mononuclear cells is reverse transcribed using antisense primers, specific for different locus (DQB, DQA, DPA or DPB) or group of loci (DRB1-5, or HLA-A and HLA-B and HLA-C). The synthesized cDNA molecules are then enzymatically amplified using different combinations of oligonucleotides for each locus and directly sequenced with Taq polymerase using an internal oligonucleotide. The sequenced genes are then analyzed.

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of HLA-A, HLA-B, and HLA-C transcripts enzymatically amplified and sequenced using a limited number of selected oligonucleotide.

DNA sequence-based HLA class I typing method

The present invention provides a process for determining genotypes in highly polymorphic systems by polymerase chain reaction amplification of cDNA or genomic DNA and direct sequencing polymerase chain reaction products using oligonucleotide primers. More specifically, Class I HLA genotypes can be unambiguously determined in any subject in 16-24 hours by direct sequencing of HLA-A, HLA-B, and HLA-C transcripts enzymatically amplified and sequenced using a limited number of selected oligonucleotides.

Growing community of inventors

Finksburg, MD, United States of America

Michael T Boyce-Jacino