Lynx Therapeutics, Inc.

Applera Corporation

Pe Corporation (ny)

Life Technologies Corporation

The Perkin-elmer Corporation

Jer-Kang Chen

Sergei M Gryaznov

Ronald G Schultz

Roger A O'Neill

Claudia Chiesa

Steven Menchen

George A Fry

Barnett B Rosenblum

Michael W Hunkapiller

Mark R Andersen

John S Shigeura

Zhaochun Ma

Paul M Kenney

Majid Mehrpouyan

Joe Y Lam

Stephen Lambert

Vergine C Furniss

Boli Huang

George Fry

Shoeb Khan

Nucleotide analogs that can sustain the enzymatic synthesis of double-stranded nucleic acid from a nucleic template are described. The nucleotide analogs include: (i) a base selected from the group consisting of adenine, guanine, cytosine, thymine, uracil and their analogs; (ii) a label attached to the base or analog of the base via a cleavable linker; (iii) a deoxyribose; and (iv) one or more phosphate groups. The linker and/or the label inhibits template directed polymerase incorporation of a further nucleotide substrate onto an extended primer strand. In addition, cleavage of the linker leaves a residue attached to the base which is not present in the natural nucleotide and which does not inhibit extension of the primer strand. The nucleotide analogs can therefore be used as reversible terminators in sequencing by synthesis methods without blocking the 3' hydroxyl group. Methods of sequencing DNA using the substrates are also described.

Unnatural polymerase substrates that can sustain enzymatic synthesis of double stranded nucleic acids from a nucleic acid template and methods of use

Apparatus and methods for separating different polynucleotide populations in a mixture are provided, wherein different polynucleotides or polynucleotide populations are captured on different solid support. After hybridization, polynucleotides are selectively released from a selected support by altering a physical property of that support. The released polynucleotides can be eluted from a common flow path and isolated.

Apparatus and method for specific release of captured extension products

Ligation-based methods and kits are disclosed for determining the degree of methylation of one or more target nucleotides. In certain embodiments, the methylation status of one or more target nucleotides is determined by generating misligation products. In certain embodiments, at least one target nucleotide is amplified prior to the ligation reaction. In certain embodiments, at least one ligation product, at least one ligation product surrogate, at least one misligation product, at least one misligation product surrogate, or combinations thereof are amplified. In certain embodiments, one or more ligation probes comprise at least one nucleotide analog, at least one Modification, at least one mismatched nucleotide, or combinations thereof.

Methods and kits for methylation detection

The present invention provides substrates and apparatuses for efficient, rapid and specific capture, and optimal recovery, of nucleic acids, as well as methods of their use. The substrate is porous in nature and has a capture polynucleotide capable of hybridizing to a target nucleic acid immobilized thereon. Upon flowing a sample containing or suspected of containing the target nucleic acid through the porous substrate, the target nucleic acid is rapidly captured. Following capture, the target nucleic acid can be efficiently recovered for subsequent use.

Method and apparatus for flow-through hybridization

The invention relates to methods and compositions for simultaneously generating a plurality of polynucleotide sequencing ladders or PCR amplification products. Each sequencing ladder is generated from a recoverable primer, i.e., an oligonucleotide primer comprising a recovery tag. The recovery tag may be an oligonucleotide. Each sequencing ladder has a unique recovery tag. After the generation of the multiple sequencing ladders, the different sequencing ladders are separated from one another, i.e., purified, by binding to recovery tag binding compounds that have been immobilized on one or more solid supports. The recovery tag binding compounds are immobilized on the solid support in an addressable manner, i.e., the recovery tag binding compounds have distinct locations on the solid support. The binding of the sequencing ladders to the recovery tag binding compounds serves to separate the different polynucleotide sequencing ladders present in a given solution. The separated sequencing ladders may then be released from the immobilized recovery tag binding compounds and subsequently resolved into individual components of the sequencing ladders or PCR products. The subject methods of separating sequencing ladders simultaneously generated in the same vessel may readily be adapted to separate a plurality of simultaneously generated polynucleotide amplification products. Other aspects of the invention are kits for the generation or recovery of a plurality of polynucleotide sequencing ladders or amplification products. The kits comprise a plurality of recoverable primers having unique recovery tags. Preferably, the recovery tags are polynucleotides that have substantially the same denaturation temperature when bound to appropriate recovery tag binding compounds, i.e., the primers comprise an integrated set. The kits may further comprise recovery tag binding compounds. The kits may further comprise labeled chain terminators. Other aspects of the invention include polynucleotide recovery devices.

Multiplex polynucleotide capture methods and compositions

Modified oligonucleotides 3&prime;-NHP(O)(O,)O-5&prime; phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Oligonucleotide N3&prime;&rarr;N5&prime;Phosphoramidate Duplexes

Oligonucleotide N3'.fwdarw.P5' phosphoramidates

Modified oligonucleotides 3'-NHP(O)(O.sup.-)O-5' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Oligonucleotide N3'-P5' phosphoramidates: hybridization and nuclease

Modified oligodeoxyribonucleotide 3'--NHP (O) (O.sup.-) O-5' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Oligodeoxyribonucleotide N3' P5' phosphoramidates

Oligonucleotide N3'.fwdarw.P5' phosphoramidates: hybridization and

Modified oligonucleotide 3'-NHP(O)(O.sup.-)O-5' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Oligonucleotide N3.fwdarw.P5' phosphoramidates: triplex DNA formation

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Palo Alto, CA, United States of America

Jer-Kang Chen