US Patent 7,388,092. Oligonucleotides and analogs labeled with energy transfer dyes
Inventors: Linda Lee, Sandra Spurgeon, Barnett Rosenblum
Describes new linkers for coupling a donor dye to an acceptor dye. The linkers facilitate the efficient transfer of energy between a donor and acceptor dye. One of them has the general structure R21Z1C(O)R22R28 where R21 is a C1-5 alkyl attached to the donor dye, C(O) is a carbonyl group, Z1 is either NH, sulfur, or oxygen, R22 is a substituent which includes an alkene, diene, alkyne, a five- and six-membered ring with at least one unsaturated bond, or a fused ring structure which is attached to the carbonyl carbon, and R28 includes a functional group which attaches the linker to the acceptor dye.
US Patent 7,387,878. Methods for DNA amplification and sequencing
Inventors: Aviva Lapidot, Robert Iakobashvili, Gennady Malin
Assignee: Yeda Research and Development
The osmoprotectants proline, 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine ("THP(B)"), and 2-methyl-4-carboxy-5-hydroxy-3,4,5,6,-tetrahydropyrimidine ("THP(A)") are capable of increasing the thermal stability of DNA polymerases at elevated temperatures. THP(B) is further effective in lowering the melting temperature of double-stranded DNA. Proline, THP(A), and THP(B) are thus useful in procedures involving melting of double-stranded DNA and/or polymerase-mediated DNA synthesis, such as in primer extension, in PCR (polymerase chain reaction) amplification, and in DNA sequencing.
US Patent 7,387,876. Amplification of trace amounts of nucleic acids
Inventors: George Church, Kun Zhang
Assignee: President and Fellows of Harvard College
Describes methods of reducing background during amplification of small amounts of nucleic acids by analyzing sources of low-level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. "Primer-dimer" background can be reduced by judicious design of primers. The inventors have shown clean signal-to-noise with as little starting material as one single human cell (about 6 picogram), E. coli cell (about 5 femtogram) or Prochlorococcus cell (about 3 femtogram).
US Patent 7,384,737. Synthesis of spatially addressed molecular arrays
Inventor: Colin Barnes
Discloses methods for forming spatially addressable arrays of polynucleotides of known sequence by using blocking groups that prevent the incorporation of multiple nucleotides during each incorporation step. The formation of spatially addressed high-density arrays has many important benefits for the study of the single polymer molecules and their interactions with other biological molecules. The arrays are particularly suitable for DNA analysis procedures using hybridization-based approaches. Knowing the sequence of polynucleotides (polymers) on the array enables the user to quickly determine the sequence of a complementary polynucleotide hybridized to it.
US Patent 7,381,529. Methods and compositions for detecting nucleic acids using scanning probe microscopy and nanocodes
Inventors: Mineo Yamakawa, Andrew Berlin
Provides a method for determining a nucleotide sequence of a nucleic acid that includes contacting the nucleic acid with a series of labeled oligonucleotides for binding to the nucleic acid. Each labeled oligonucleotide includes a known nucleotide sequence and a molecular nanocode. The nanocode of an isolated labeled oligonucleotides that binds to the nucleic acid is then detected using scanning probe microscopy. Nanocodes of the invention, in certain aspects, include detectable features beyond the arrangement of tags that encode information about the barcoded object, which assist in detecting the tags that encode information about the barcoded object. The detectable features include structures of a nanocode or associated with a nanocode for error checking and error-correction, encryption, and data reduction and compression.
US Patent 7,378,242. DNA sequence detection by limited primer extension
Inventor: Richard Hurt
Assignee: Atom Sciences
A novel limited primer extension reaction improves detection sensitivity and specificity in a variety of hybridization platforms. In the invention, a sequence of target DNA that lacks one of the four types of nucleic acid bases for a span of eight or more adjacent nucleotide positions is selected for use. This sequence is referred to as the extension complement sequence, or ECS. A primer with a sequence that is complementary to the target sequence that is immediately downstream to the 3' side of this ECS is used to initiate an extension reaction. Extension occurs using a DNA polymerase and standard deoxynucleoside triphosphates for three of the four types of nucleic acid bases. The fourth base, which is complementary to the base missing in the ECS, is either absent or present only in the form of a dideoxynucleoside triphosphate, which does not support further extension. In either case, the extension reaction does not proceed past the first occurrence in the template of the base that is missing in the ECS. This results in a primer extension product with fixed length determined by the length of the ECS. The process can be repeated using a thermal-stable polymerase in a thermal-cycled reaction that results in a linear amplification of the targeted sequence. The resulting limited primer extension products serve as ideal hybridization analytes for determination of sample sequence content using microarrays.
US Patent 7,374,885. Single-primer nucleic acid amplification methods
Inventors: Michael Becker, Wai-Chung Lam, Kristin Livezey, Steven Brentano, Daniel Kolk, Astrid Schroder
The invention is directed to novel methods of synthesizing multiple copies of a target nucleic acid sequence which are autocatalytic, i.e. able to cycle automatically without the need to modify reaction conditions such as temperature, pH, or ionic strength, and using the product of one cycle in the next one. In particular, it discloses a method of nucleic acid amplification that is robust and efficient, while reducing the appearance of side products. The method uses only one primer, the "priming oligonucleotide," a promoter oligonucleotide modified to prevent polymerase extension from its 3'-terminus and, optionally, a means for terminating a primer extension reaction, to amplify RNA or DNA molecules in vitro, while reducing or substantially eliminating the formation of side products.
US Patent 7,374,882. Method for base sequencing and biologically active nucleic acids
Inventor: Yoshihide Hayashizaki
Aptamers are nucleic acids and similar molecules, such as peptide-nucleic acids, that specifically bind to a ligand such as a protein or peptide. The present invention provides aptamers comprising at least one base capable of base-pairing and different from the standard Watson-Crick bases. The invention also relates to a method for preparation of such aptamers and to methods for sequencing nucleic acids that comprise at least one base capable of base pairing and different from the standard Watson-Crick bases.
US Patent 7,374,881. Method for collecting and using nuclear mRNA
Inventor: Masato Mitsuhashi
Assignee: Hitachi Chemical Research Center
The invention is related to a novel method of detecting mRNA, based on the fact that the newly synthesized mRNA is confined inside the cell nucleus. Cells are captured on a membrane and then treated by a cell membrane permeation solution, thereby increasing permeability of cell membranes. After the cytoplasm is washed away, nuclei are dissolved with a cell dissolving solution, and mRNA can be recovered from the obtained solution.