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Sequencing-Related US Patents Granted Aug. 6 – Sept. 2, 2008

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US Patent 7,419,833. Method for nucleic acid sequencing
Inventor: Kuniaki Nagayama 
Assignee: Nagayama IP Holdings
 
Describes a method for determining the sequence of at least a portion of a single-stranded nucleic acid molecule by base-specifically labeling the exposed bases using heavy element-substituted nucleotide bases which form Watson-Crick-type base pairs with the exposed bases and then imaging the labeled single-stranded nucleic acid molecule using electron microscopy, e.g., transmission electron microscopy, or some other method that permits discrimination of the heavy element substituted nucleotide bases. The image is analyzed to determine the base sequence of at least a portion of the nucleic acid molecule.
 

 
US Patent 7,410,564. Apparatus and method for biopolymer identification during translocation through a nanopore
Inventor: Curt Flory
Assignee: Agilent Technologies
 
Provides an apparatus and method for identifying and sequencing a biopolymer translocating a nanopore. The apparatus of the invention provides a first electrode, a second electrode, and a potential means for applying a bias ramping potential across the electrodes to produce resonant tunneling of current carriers between the two electrodes. As the bias potential is ramped across the electrodes, the increase in tunneling current occurs as the carrier energy sequentially matches the conduction band energies of the translocating biopolymer. This technique allows for real-time identification and sequencing of a biopolymer as the band energy spectra of the individual portions of the bipolymer are recorded, differentiated, and identified. A method for identifying the biopolymer is also disclosed.
 

 
US Patent 7,416,845. pH dependent ion exchange material, solid substrate having the material immobilized on its surface, and method of isolating a nucleic acid using the material or the solid substrate
Inventor: Jong-Myeong Park
Assignee: Samsung Electronics
 
Provides a pH-dependent ion exchange material with a carboxyl group, an amino group, and a polyethylene oxide moiety, which is used for isolating a nucleic acid. Also provides a method of isolating a nucleic acid using the material or the solid substrate. The pH-dependent ion exchange material has at least two monomers selected from a group of monomers.
 

 
US Patent 7,416,844. Composition for nucleic acid sequencing
Inventors: Jonas Korlach, Watt Webb, Michael Levene, Stephen Turner, Harold Craighead, Mathieu Foquet
Assignee: Cornell Research Foundation
 
The invention is directed to a method of sequencing a target nucleic acid molecule with several bases. The temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. Several labelled types of nucleotide analogs are provided to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.
 

 
US Patent 7,414,118. Modified oligonucleotides and applications thereof
Inventors: Khairuzzaman Bashar Mullah, Zhaochun Ma
Assignee: Applied Biosystems
 
Disclosed, among other things, are primers containing certain modified nucleobases in the 3'-terminal region of the primers that provide reduced formation of primer-dimers during amplification reactions, and various methods of their use.
 

 
US Patent 7,414,116. Labelled nucleotides
Inventors: John Milton, Silke Ruediger, Xiaohai Liu 
Assignee: Illumina Cambridge
 
The invention provides a nucleotide or nucleoside with a base attached to a detectable label via a cleavable linker, characterized in that the cleavable linker contains a moiety selected from the group comprising a formula wherein X is selected from the group comprising O, S, NH, and NQ, wherein Q is a C1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH, and N(allyl), T is hydrogen or a C1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside.
 

 
US Patent 7,413,862. FRET probes and methods for detecting interacting molecules
Inventors: Jacobus Johannes Maria van Dongen, Frank Jakob Theodor Staal
Assignee: Erasmus Universteit Rotterdam
 
The invention relates to the field of detecting interacting molecules. It provides a set of a first and a second probe, each such probe provided with a dye allowing energy transfer; at least one probe provided with a reactive group allowing juxtaposing the first and second probe. A method is provided for detecting at least two interacting molecules at the single-cell level using a set of probes.
 

 
US Patent 7,413,855. Method for bisulfite treatment
Inventors: Frank Bergmann, Christine Markert-Hahn, Joerg Kleibler, Dirk Block
Assignee: Roche Molecular Systems
 
The application is directed to a method for performing a bisulfite reaction to determine methylation positions in a nucleic acid, i.e. methylated and non-methylated cytosines. The nucleic acid is incubated in a solution for a time period of 1.5 to 3.5 hours at a temperature between 70 and 90 degrees Celsius, whereby the concentration of bisulfite in the solution is between 3 M and 6.25 M and whereby the pH value of the solution is between 5.0 and 6.0. The nucleic acid, i.e. the cytosine bases in the nucleic acid, are deaminated. Then the solution comprising the deaminated nucleic acid is desulfonated and preferably desalted. The application is further related to a solution comprising bisulfite with a certain pH and its uses as well as a kit comprising the solution.
 

 
US Patent 7,413,708. Methods and reagents for combined PCR amplification
Inventor: Paul Mayrand
Assignee: Applied Biosystems
 
An oligonucleotide probe is disclosed, which includes an oligonucleotide, a fluorescent molecule attached to one of its ends, and a quencher molecule attached to the opposite end. The probe is rendered impervious to digestion by the 5'-3' exonuclease activity of a polymerase and the 5'-3' extension by a polymerase. The invention also includes methods for performing combined PCR amplification and hybridization probing, one such method including the steps of contacting a target nucleic acid sequence with PCR reagents and an oligonucleotide probe as described above, and subjecting these reagents to thermal cycling. One preferred refinement of the method further includes the addition of a strand displacer to facilitate amplification.
 

 
US Patent 7,410,782. Thermostable enzyme promoting the fidelity of thermostable DNA polymerases — for improvement of nucleic acid synthesis and amplification in vitro
Inventors: Waltraud Ankenbauer, Frank Laue, Harald Sobek, Michael Greif 
Assignee: Roche Diagnostics, Roche Molecular Systems
 
A purified thermostable enzyme is derived from the thermophilic archaebacterium Archaeoglobus fulgidus. The enzyme can be native or recombinant, is stable under PCR conditions, and exhibits double-strand-specific exonuclease activity. It is a 3'-5' exonuclease and cleaves to produce 5'-mononucleotides. Thermostable exonucleases are useful in many recombinant DNA techniques, in combination with a thermostable DNA polymerase like Taq especially for nucleic acid amplification by the polymerase chain reaction.
 

 
US Patent 7,410,760. Controls for primers in multiplex amplification reactions
Inventor: David Swenson 
Assignee: Cepheid
 
The invention provides methods and compositions for confirming the integrity of primers and other components of amplification reactions, including multiplex amplification reactions.

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