Marker Gene Monthly Newsletter - Volume 11, Number 11 - November, 2011

ASCB Cell Biology Meeting 2011

Marker Gene will be exhibiting at the 51st Annual Meeting of the American Society for Cell Biology in Denver, CO December 3rd-6th, 2011. Please stop by and visit our booth (# 424) or come to one of our poster presentations on some of our new assay developments. Details about these posters are given below. We will also be introducing several other new products for live cell analysis techniques at the meeting. For more information please see the ASCB website, and the presentations shown below or visit our website.

Please stop by to discuss your research and development efforts. For updates or to schedule a meeting with our staff please visit our website or email us directly at techservice@markergene.com.  We appreciate your input for new products and methods that are of interest to the worldwide scientific community. We can also develop products for your specific applications and for exclusive uses.

For more information about any of our products, simply telephone us toll free at 1-888-218-4062 or contact us by e-mail at techservice@markergene.com. We will be happy to send you more details about our existing and new products and their specifications.

Cell Biology Assays Using Improved DNA Detection Reagents.

Monday, December 5 12:30 PM– 2 PM 1156/B607

Symmetrical homodimers of cyanine dyes are known to have exceptional sensitivity for detection of nucleic acids with very high fluorescence enhancement upon binding to DNA. In addition, they exhibit increased quantum yields and stability upon binding due to their high affinity for the nucleic acids. We have recently synthesized a series of homodimeric dyes including 6-chloro-YOYO-1 and 6-chloro-TOTO-1 that are analogs of the commercial dyes YOYO-1 and TOTO-1. They differ from the commercial products in that they have chloro-substituents at the six-position of the benzoxazole or benzothiazole rings respectively. Each dye has also been prepared with different anionic counterions imparting improved water solubility. In addition, their excitation and emission spectra upon DNA binding (EX 450 and 520 nm; EM 510 - 540 nm, quantum, yield 0.5) overlaps well with common fluorescence detection systems.
These new analogs have been found to have an approximately two-fold increased sensitivity in agarose gel electrophoresis detection of dsDNA prestained with these new dyes over their parent analogs. The new dyes have found application in a number of cell biology assays. Staining of fixed chromatin of human breast cancer cells (MDA-MB-231) exhibited bright green signal, providing an alternate fluorescence detection of chromatin/DNA over DAPI. The new dyes have also found use for post-staining amplification products in Loop Mediated Isothermal Amplification (LAMP) assays. In this assay, we utilized 4 primer sequences which recognize 6 distinct regions of bacteriophage DNA and a strand disrupting polymerase for amplification under isothermic conditions. In addition, upon application at 1 uM concentration, the new dyes have been found to be suitable for staining flash frozen, ethanol fixed brain tissue sections. Since these new dyes are cell-membrane impermeant, they have been found to be suitable for distinguishing dead cells when used at a 0.5 uM and therefore act as a vital reagent in Live:Dead format assay. In addition, they are less hazardous than other DNA stains and can thus be utilized in live cell analysis formats. In conclusion, our new 6-chloro-YOYO-1 and 6-chloro-TOTO-1 dyes have increased sensitivity in detection and can be a suitable reagent for a number of important biological assays. This work is funded in part by grant number NSF-IIP0923953 from the National Science Foundation-USA

• Harlan F, Jiang Y, Galvao R, Deligeorgiev T, Naleway JJ (2011) "Cell Biology Assays Using Improved DNA Detection Reagents." Presented at the American Society for Cell Biology Meeting, Denver, CO, December, 2011 Poster 1156/B607.
  

Toward Unbiased Direct DNA and RNA Labeling Systems.

Monday December 5 2PM– 3:30 PM 1157/B608

Problems with current genomic microarray analysis techniques include hybridization perturbation caused by base labeling and enzyme-introduced sequence bias for labeling. To solve these problems, we have developed a series of new ultrasensitive labeling reagents for directly labeling DNA or RNA samples isolated from live cells or tissues or prepared by PCR. From their unique diazo reactive group as well as 31P-NMR studies, these labeling reagents have been found to directly modify terminal and backbone phosphate groups of DNA or RNA samples. We developed these new reagents for use in fluorescent (TAMRA, Cy3, Coumarin) as well as hapten (biotin) labeling protocols. The new labeling reagents have been shown to cause less hybridization perturbation.
The effects of the direct labeling method on hybridization efficiency as well as in comparison with existing techniques were examined. Biotin labeling of miRNA isolated from NIH3T3 murine fibroblasts was hybridized to Affymetrix GeneChip miRNA (v1) arrays and compared to poly(A) polymerase enzyme-based labeling which is the adopted labeling method used in most miRNA microarray protocols. We observed a 0.68 Pearson Correlation Coefficient between these two labeling methods, with the new direct labeling reagent detecting 24.4% more sequences. A 13.3% bias in labeling was found for sequences ending with a 3’-uracil for the enzymatic labeling method versus a 1.7% bias for our chemical labeling method. Certain hairpin sequences also exhibited biased labeling. Reports from multiple labs regarding enzymebased bias in labeling are consistent with these observations. To confirm the differential labeling is due to the enzymatic sequence bias, we also compared biotin labeled miRNA with a chloroethylamino reactive biotin direct-labeling reagent which directly modifies the bases of nucleotides. We observed a 0.97 Pearson Correlation Coefficient with 17.8% higher mean intensity using our new direct labeling over the chloroethylamino direct labeling biotin system. In addition, all four miRNA probes miR-{467a,328,206,214}, which lack G,A,C and U respectively, were labeled and exhibited minimal sequence dependence compared with a 10% hybridization bias for the reactive chloroethylamino biotin labeling of miR-328 and 214 over miR-467a and 206. We also isolated miRNA from both etoposide-treated and non-treated NIH3T3 cells, and labeled them using our direct biotin labeling reagent and analyzed the miRNA patterns using the miRNA Affymetrix (v1) arrays. The differential expression miRNA patterns obtained upon etoposide treatment were consistent with the literature as well as confirmed by northern blot and qPCR. Uses of these methods to determine the pattern of gene expression upon therapeutic treatment as well as for pharmacokinetic analysis of DNA or RNA based drug therapies have been developed. This work is supported by NSF grant IIP-0923953.

• Jiang Y, Harrington CA, Naleway MA, Strain SM, Savage SL, Slottke RE, Vartanian KA, Naleway JJ (2011) "Toward Unbiased Direct DNA and RNA Labeling Systems." Presented at the American Society for Cell Biology Meeting, Denver, CO, December, 2011 Poster 1157/B608
  

Next Generation Red Luciferase Vector.

Marker Gene has a well established line of vectors containing a new red luciferase gene developed by utilizing point mutation and codon optimization of the luciferase sequence from the natural luciferase gene isolated from Luciola cruciata (the Japanese firefly). This novel recombinant DNA has been incorporated into vectors (M1394 and M1395) containing the highly expressing CMV and SV40 promoters for high expression activities in mammalian cells. Marker Gene is now excited to launch our next generation red luciferase vector: pFHluc (M1796). This vector retains the original features of the M1394 vector such as high expression under the CMV promoter and increased thermostability but also includes the additional functionality of a multiple cloning site containing 11 unique restriction sites. This cloning site will allow genes to be easily coexpressed with luciferase within mammalian cells.

This novel luciferase gene codes for an improved amino acid sequence which exhibits long-wavelength light emission (red color, EM 619 nm), as well as improved thermostability and higher expression levels in mammalian cells compared with other luciferases including the native luciferase derived from the American firefly Photinus pyralis. The expressed enzyme uses the same substrate, D-Luciferin (M0237), that is used with other luciferases, and activity can easily be detected using our convenient MarkerGeneTM Live Cell Luciferase Assay Kit (M0626). Additional substrates for use in detecting cloned activity in living tissues and in vivo include the membrane permeant D-Luciferin, ethyl ester (M0906). These features along with the multiple cloning site makes this new vector an ideal candidate for use in vivo detection methods of cloned luciferase activity. It can also be used concurrently with vectors such as the pGL3 Photinus pyralis vectors for multiplexed detection of two cloning events in the same cell line. Transfection into eukaryotic cells may be mediated by cationic lipid compounds like Lipofectamine™, TransIt® calcium phosphate, DEAE-dextrans, or electroporation. The modified luciferase protein is also available for use in coupled enzyme assays, high-sensitivity detection methods and general enzymology uses. For more information about these systems, please see the references below or visit our website.

• Coleman DJ, Naleway JJ, Cook GM (2010) "Modified Luciola Cruciata Luciferase Gene and Protein." US Patent 7,723,502.
• Coleman, DJ, McGibney, MT, Cook, GM, Naleway, JJ (2008) "Mammalian Luciferase Decection Vectors and Applications." Experimental Biology Meeting, San Diego, CA April, 2008, Abstract No. 1028.1. link
• Tatsumi H, Masuda T, Kajiyama N, Nakano E (1989) "Luciferase cDNA from Japanese firefly, Luciola cruciata: cloning, structure and expression in Escherichia coli." J. Biolumin. Chemilumin. 3(2):75-78.
• Masuda T, Tatsumi H, Nakano E (1989) "Cloning and sequence analysis of cDNA for luciferase of a Japanese firefly, Luciola cruciata." Gene. 77(2):265-270.
• de Wet JR, Wood KV, Helinski DR, DeLuca M (1985) "Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli." Proc Natl Acad Sci U S A. 82(23):7870-7873.
• Mamaev SV, Laikhter AL, Arslan T, Hecht SM, (1996) "Firefly Luciferase: Alteration of the Color of Emitted Light Resulting from Substitutions at Position 286."
• Kajiyama, N, Nakano E, (1991) "Isolation and characterization of mutants of firefly luciferase which produce different colors of light." Protein Eng. 4: 691.
• Kajiyama N, Nakano E, (1993) "Thermostabilization of Firefly Luciferase by a Single Amino Acid Substitution at Position 217." Biochemistry 32: 13795-1 3799.

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CONTRACT RESEARCH @ markergene.com

• Established in 1993 at the University of Oregon Riverfront Research Park.
• Screening Assay Development for HTS and uHTS
• Chemical and Cellular Assays - High-Content Screening.
• DNA/RNA (genomics) and protein (proteomics) labeling and assay development.
• Pharmaceutical Intermediates - design, synthesis, and in vitro testing in mammalian cell culture.
• Specializing in Carbohydrate, Lipid, Peptide, and Nucleic Acid Chemistries.
• Fully equipped laboratories (Biochemistry, Chemical Synthesis, Tissue Culture, Analytical).
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