Showing papers by "Stratagene published in 2004"

Universal Reference RNA as a standard for microarray experiments

Abstract: Obtaining reliable and reproducible two-color microarray gene expression data is critically important for understanding the biological significance of perturbations made on a cellular system. Microarray design, RNA preparation and labeling, hybridization conditions and data acquisition and analysis are variables difficult to simultaneously control. A useful tool for monitoring and controlling intra- and inter-experimental variation is Universal Reference RNA (URR), developed with the goal of providing hybridization signal at each microarray probe location (spot). Measuring signal at each spot as the ratio of experimental RNA to reference RNA targets, rather than relying on absolute signal intensity, decreases variability by normalizing signal output in any two-color hybridization experiment. Human, mouse and rat URR (UHRR, UMRR and URRR, respectively) were prepared from pools of RNA derived from individual cell lines representing different tissues. A variety of microarrays were used to determine percentage of spots hybridizing with URR and producing signal above a user defined threshold (microarray coverage). Microarray coverage was consistently greater than 80% for all arrays tested. We confirmed that individual cell lines contribute their own unique set of genes to URR, arguing for a pool of RNA from several cell lines as a better configuration for URR as opposed to a single cell line source for URR. Microarray coverage comparing two separately prepared batches each of UHRR, UMRR and URRR were highly correlated (Pearson's correlation coefficients of 0.97). Results of this study demonstrate that large quantities of pooled RNA from individual cell lines are reproducibly prepared and possess diverse gene representation. This type of reference provides a standard for reducing variation in microarray experiments and allows more reliable comparison of gene expression data within and between experiments and laboratories.

DNA polymerase fusions and uses thereof

Abstract: The present invention discloses methods of using DNA polymerase fusions at high pH in PCR, DNA sequencing and mutagenesis protocols.

Compositions and methods using dendrimer-treated microassays

Abstract: The present invention provides a chemically reactive surface able to covalently react with substances containing a hydroxyl group and/or amine group, comprising a solid surface having an activated dendrimer polyamine covalently bonded to said surface through a silane containing reagent, wherein the dendrimer polyamine can covalently bind the substance comprising a hydroxyl group and/or amino group. The present invention further provides a method for producing chemically reactive surfaces for binding moieties comprising a hydroxyl group and/or amine group, as well as kits comprising the chemically reactive surface of the invention.

Compositions and methods for protein isolation

Abstract: The invention provides for polynucleotides and vectors comprising at least two tag sequences. The invention also provides for polynucleotides and vectors comprising a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for polynucleotides and vectors wherein a gene of interest is fused in frame to at least two tag sequences, for example, a streptavidin binding peptide sequence and a calmodulin binding peptide sequence. The invention also provides for the chimeric proteins encoded by these polynucleotides. The invention also provides for methods of using the polynucleotides of the invention for detecting and/isolating protein complexes or identifying a binding partner for a protein of interest.

Compositions and kits for Herpes Simplex Virus type 1 and 2 nucleic acid detection

Abstract: The invention relates to polynucleotides for HSV detection and the use of these polypeptides in kits and methods for HSV detection.

Compositions and kits for detection of a nucleic acid by sequential amplification

Abstract: The invention relates to nucleic acid compositions for use in sequential amplification detection assays which permit the detection of a nucleic acid target in a sample.

Methods and compositions for the detection of bacterial species

Abstract: The invention relates to compositions, methods and kits for the PCR-based detection of bacterial species. Methods are provided for increasing the specificity of a PCR-based bacterial assay. More specifically, primer sets and PCR-based assays are provided that amplify and detect conserved 16S rRNA gene sequences from multiple Mycoplasma species. The invention also provides kits for performing those assays, and compositions comprising oligonucleotide primers useful in those assays.

Nucleic acids, compositions, methods, and kits for detecting mycoplasma and acholeplasma species

Abstract: The invention relates to nucleic acids, methods, compositions, and kits for the PCR-based detection of Mycoplasma and Acholeplasma bacterial species. The nucleic acids, methods, compositions, and kits provide for increased specificity and sensitivity of PCR-based Mycoplasma bacterial assays. Primer sets and PCR-based assays are provided that amplify and detect conserved 16S rRNA gene sequences from multiple Mycoplasma and Acholeplasma species while avoiding amplification and detection of non-target sequences.

Renilla gfp mutants with increased fluorescent intensity and special shift

Abstract: The present invention provides a polynucleotides encoding mutants of green fluorescent protein from Renilla reniformis, including humanized sequences which permit enhanced expression of the encoded polypeptides in mammalian cells.

Apparatus and method for flexible heating cover assembly for thermal cycling of samples of biological material

Abstract: A flexible heating cover assembly for an apparatus for heating samples of biological material with substantial temperature uniformity includes a housing having a plurality of engageable enclosure components; a resistive heater having a plurality of heater element areas; a heater backing plate providing stability to the resistive heater; a force distribution system that distributes a force over the heater backing plate; and a support plate providing stiffness for the force distribution system, wherein the arrangement of the resistive heater, the heater backing plate, the force distribution system and the support plate provide substantial temperature uniformity among a plurality of sample tubes for receiving samples of biological material. The flexible heating cover assembly improves the uniformity, efficiency, quality, reliability and controllability of the thermal response during thermal cycling of the biological material.

Methods and compositions for high level expression of a heterologous protein with poor codon usage

Abstract: High level expression of heterologous proteins in host cells is frequently limited by the presence of rarely utilized codons, due to depletion of the internal tRNA pool and stalling of translation. This invention provides expression host cells generated by introduction of a vector for the expression of an array of tRNA genes that are rare in the host cells. The modified host cells are capable of efficiently supporting expression of selected recombinant genes which otherwise would be limited by the presence of rare codons.

Compositions and methods for polynucleotide sequence detection

Abstract: The present invention provides compositions, kits, and methods for detecting polynucleotide sequence differences. The method involves amplifying a polynucleotide in the presence of a labeled nucleotide whose incorporation into the amplified product can indicate the presence of a sequence difference within the polynucleotide template. The invention is particularly useful for differentiating two or more closely related polynucleotide sequences, for example, in determining which allele or alleles of a multiallelic organism are present in a target polynucleotide.

Pfu replication accessory factors and methods of use

Abstract: This invention provides isolated polynucleotides that encode replication accessory factors. The invention also provides novel DNA replication accessory factors, which have been isolated and purified from the hyperthermophilic archaea Pyrococcus furiosus. The invention also provides various methods of enhancing a nucleic acid polymerase reaction comprising the addition of the replication accessory factors to the reaction. This invention further provides methods of synthesizing, amplifying, and mutagenizing nucleic acids of interest employing the replication accessory factors. This invention also provides kits comprising at least one of the replication accessory factors. This invention also provides kits useful for various methods that comprise at least one replication accessory factor.

Electrocompetent host cells

Abstract: Provided are methods of generating electrocompetent bacterial cells, the methods involving the growth of the cells at hyperosmotic salt concentration. Also provided are methods of producing a transformed cell, methods of producing a recombinant polypeptide, and strains of E. coli that exhibit increased electrotransformation efficiency.

Compositions and methods utilizing stable reporter cell lines for detection of pathway-specific signal transduction

Abstract: The invention encompasses compositions and methods which utilize a cell line comprising a stably integrated recombinant nucleic acid construct comprising a reporter gene operably lined to a recognition sequence for a sequence-specific DNA-binding protein and a stably integrated recombinant nucleic acid construct comprising a sequence encoding a fusion protein, the fusion protein comprising a sequence-specific DNA binding domain, wherein the DNA binding domain specifically binds the recognition sequence, and a conditionally active transactivation domain, wherein activation of the conditionally active transactivation domain is dependent on protein phosphorylation and/or protein: protein interaction, and wherein binding of the fusion protein to the recognition sequence results in transactivation of the reporter gene expression when the transactivation domain fused to the DNA binding domain is activated.