Lynx Therapeutics, Inc.

Other

Gilead Sciences, Inc.

Solexa, Inc.

The Molecular Sciences Institute, Inc.

Glenn Albrecht

Sydney Brenner

Robert B Dubridge

Stephen C Macevicz

Sergei M Gryaznov

Sarah N McCurdy

Michael C Pallas

David H Lloyd

Rongdian Fu

John J Toole

Linda C Griffin

John A Latham

Lawrence L Leung

Eric Vermaas

Disclosed are methods for identifying nucleic acid sequences which are of different abundances in different nucleic acid source populations, e.g. differentially expressed genes or genomic variations among individuals or populations of individuals. In one embodiment, probes derived from the source nucleic acid populations are derivatized with a terminal sample ID (SID) sequence characteristic of that population. Upon competitive hybridization of the probes to a reference or index nucleic acid library containing all the sequences in the populations being compared, the SID tags remain single stranded, and those from the different sources are then annealed to one another. Unhybridized (remainder) SID sequences are then quantified. By labeling such remainder SID sequences with a fluorescent dye, FACS sorting of beads containing the hybridized probes can be carried out. The signal ratio upon which such sorting is based is enhanced compared to competitive hybridization using labeled probes without SID sequences.

Method for determining relative abundance of nucleic acid sequences

The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluorescent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. The invention also provides a method for identifying and isolating rare genes. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.

Solid phase selection of differentially expressed genes

The invention provides a method and materials for monitoring and isolating differentially expressed genes. In accordance with the method of the invention, differently labeled populations of DNAs from sources to be compared are competitively hybridized with reference DNA cloned on solid phase supports, e.g. microparticles, to provide a differential expression library which, in the preferred embodiment, may be manipulated by fluorescence-activated cell sorting (FACS). Monitoring the relative signal intensity of the different fluoresent labels on the microparticles permits quantitative analysis of expression levels relative to the reference DNA. Populations of microparticles having relative signal intensities of interest can be isolated by FACS and the attached DNAs identified by sequencing, such as with massively parallel signature sequencing (MPSS), or with conventional DNA sequencing protocols.

The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

Oligonucleotide tags for sorting and identification

An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5&prime; strands and the use of adaptors having a 3&prime; blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3&prime; blocking groups are removed, the adjacent 5&prime; hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.

Adaptor-based sequence analysis

The invention provides a method of tracking, identifying, and/or sorting classes or subpopulations of molecules by the use of oligonucleotide tags. Oligonucleotide tags of the invention comprise oligonucleotides selected from a minimally cross-hybridizing set. Preferably, such oligonucleotides each consist of a plurality of subunits 3 to 9 nucleotides in length. A subunit of a minimally cross-hybridizing set forms a duplex or triplex having two or more mismatches with the complement of any other subunit of the same set. The number of oligonucleotide tags available in a particular embodiment depends on the number of subunits per tag and on the length of the subunit. An important aspect of the invention is the use of the oligonucleotide tags for sorting polynucleotides by specifically hybridizing tags attached to the polynucleotides to their complements on solid phase supports. This embodiment provides a readily automated system for manipulating and sorting polynucleotides, particularly useful in large-scale parallel operations, such as large-scale DNA sequencing, mRNA fingerprinting, and the like, wherein many target polynucleotides or many segments of a single target polynucleotide are sequenced simultaneously.

Kits for sorting and identifying polynucleotides

The invention provides a method of nucleic acid sequence analysis based on the ligation of one or more sets of encoded adaptors to the terminus of a target polynucleotide. Encoded adaptors whose protruding strands form perfectly matched duplexes with the complementary protruding strands of the target polynucleotide are ligated, and the identity of the nucleotides in the protruding strands is determined by an oligonucleotide tag carried by the encoded adaptor. Such determination, or 'decoding' is carried out by specifically hybridizing a labeled tag complement to its corresponding tag on the ligated adaptor.

Massively parallel signature sequencing by ligation of encoded adaptors

An improvement in adaptor-based sequence analysis is provided that addresses the problems created by self-ligation of target polynucleotides that have complementary ends. The improvement includes preparation of target polynucleotides with dephosphorylated 5' strands and the use of adaptors having a 3' blocking group. In a preferred embodiment, adaptors are ligated to target polynucleotides by a single strand, 3' blocking groups are removed, the adjacent 5' hydroxyl of the target polynucleotide is phosphorylated, and the ligation of the adaptor is completed by treatment with a ligase.

A method for identifying oligomer sequences, optionally comprising modified base, which specifically bind target molecules such as serum proteins, kinins, eicosanoids and extracellular proteins is described. The method is used to generate aptamers that bind to serum Factor X, PDGF, FGF, ICAM, VCAM, E-selectin, thrombin, bradykinin, PGF2 and cell surface molecules. The technique involves complexation of the target molecule with a mixture of oligonucleotides containing random sequences and sequences which serve as primer for PCR under conditions wherein a complex is formed with the specifically binding sequences, but not with the other members of the oligonucleotide mixture. The complex is then separated from uncomplexed oligonucleotides and the complexed members of the oligonucleotide mixture are recovered from the separated complex using the polymerase chain reaction. The recovered oligonucleotides may be sequenced, and successive rounds of selection using complexation, separation, amplification and recovery can be employed. The oligonucleotides can be used for therapeutic and diagnostic purposes and for generating secondary aptamers.

Aptamers specific for biomolecules and methods of making

The subject invention relates to a launcher and one or more gliders for use therewith. The dihedral of the glider is achieved with two parallel creases that run front to back down the center of the glider. At the leading edge of the wing surfaces, self adhesive strips of flat magnetic sheet stock can be applied to the top and bottom of each wing to provide both proper weight to initiate desirable trim to the glider and to provide a force to separate the nested planes after launching, i.e., magnetic repulsion. For a multiple launch, the gliders are stacked on top of one another and placed upside down and mounted on a launching stick. Through use of a vertical upward force the gliders are launched with sufficient force so that wind resistance will induce the glider wings to collapse upon themselves, facilitating a higher launch. At the peak of the launch, the combination of magnetic repulsion and the spring back of the glider wings to their designed high glide ratio configuration, permits each glider to successfully separate and glide to earth.

Glider with launching system

A swimming device designed to more easily facilitate the ingestion of air during the act of swimming is disclosed. The device permits the user to expend less effort breathing and focus more attention to the motor activity. The device improves the existing art of recreational snorkeling devices since its design is less cumbersome, more hydrodynamically streamlined, and causes less fatigue to the mouth and jaw muscles. The device of the invention consists of two flexible breathing tubes which encircle the user's head from the mouth, pass below the ears, join and pass through an adjustable slip piece, and then project upward and forward above the rear portion of the user's head to permit air induction free of water. At the interface of the mouth of the user, the breathing tubes are joined by a mouthpiece which has a small ribbed tube for the user to hold the device with his teeth. The device when properly tensioned with the adjustable slip piece provides a snug and self holding fit which requires no clenching of teeth or lips to maintain the position of the device. When the device is loosened by moving the slip piece along the tubes away from the mouthpiece, it easily stores around the user's neck and is independent of swim goggles or a snorkeling mask.

Growing community of inventors

Redwood City, CA, United States of America

Glenn Albrecht