Marker Gene Monthly Newsletter - Volume 11, Number 3 - March, 2011

OliGlo^TM Nucleic Acid Labeling Kits.

The ability to label DNA and RNA is important in the diagnosis and study of genetic abnormalities. A number of agents have been described for labeling nucleic acids to facilitate detection of target DNA or RNA sequences. It is essential that the labeling method not perturb base-pairing hybridization in order to preserve assay specificity. Nevertheless, several common methods have been found to lead to sequence dependent interference with the subsequent hybridization detection step. Marker Gene's new OliGlo™ kits utilize a direct labeling methodology through reaction with the phosphate groups (terminal and backbone) on oligonucleotide, DNA or RNA samples, leaving the base pairing intact for hybridization. Moreover, the active labeling reagents are prepared in situ from stable precursor molecules derived from a variety of highly fluorescent dyes or hapten labels, allowing the highly reactive labels to function at optimum efficiency for each sample. The OliGlo™ kits allow molecular biologists and clinical researchers to label and monitor genomic DNA, ssDNA or mRNA, miRNA, nucleotides or oligonucleotides for easy detection and quantification. Areas of application include general detection of target DNA/RNA/micro RNA as in Southern/northern blot, fluorescence in situ hybridization, array-based expression profiling etc. Currently available are the Universal Labeling Kits as well as  FISH kits. To find out more about these methods, please see the references below or visit our website.

• DeRisi JL, Iyer VR, Brown PO. (1997) "Exploring the metabolic and genetic control of gene expression on a genomic scale." Science 278: 680-686.,/li.
• Durrant I, Brunning S, Eccleston L, Chadwick P, Cunningham M. (1995) "Fluorescein as a label for non-radioactive in situ hybridization." Histochem. J. 27:94-99.
• Forster AC, McInnes JL, Skingle DC, Symons RH. (1985) "Non-radioactive hybridization probes prepared by the chemical labelling of DNA and RNA with a novel reagent, photobiotin." Nucleic Acids Res. 13: 745-791.
• Hoevel T, Kubbies M. (2002) "Nonradioactive labeling and detection of mRNAs hybridized onto nucleic acid cDNA arrays." Methods Mol. Biol. 185: 417-23.
• Kessler C. (1995) "Methods for nonradioactive labeling of nucleic acids." in Nonisotopic probing, blotting, and sequencing. (Kricka L J, Ed.) pp 41-109, Academic Press, San Diego.
• Olejnik J, Krymanska-Olejnik E, Rothschild KJ. (1998) "Photocleavable aminotag phosphoramidites for 5'-termini DNA/RNA labeling." Nucleic Acids Res. 26(15): 3572-3576.
• Rihn H, Coulais C, Bottin MC, Martinet N. (1995) "Evaluation of non-radioactive labelling and detection of deoxyribonucleic acids: Part one: chemiluminescent methods." Biochem. Biophys. Methods 30:91-102.

New Intercalating Dyes.

Rapid and sensitive quantitative detection of DNA is important for many medical, biological and biotechnological applications, including PCR analysis, DNA sequencing, DNA library development, agarose gel and capillary electrophoresis staining, DNA damage detection, purification of DNA fragments for subcloning, identification of DNA contaminants in recombinant protein products and in flow cytometric evaluation of biological activity. Because the intrinsic emission of nucleic acids is weak, extrinsic fluorescent probes and labels are often used for DNA staining. A wide range of intercalating fluorophores, including ethidium bromide, Hoechst 33258, acridine orange, proflavine (acridine-3,6-diamine) and DAPI (4',6-diamidino-2-phenylindole) have been employed to fluorescently stain singly stranded and doubly stranded DNA. However, these reporter molecules often suffer from background emission signals or poor enhancement of fluorescence upon binding to DNA.

Recently, the laboratory of Dr Todor Deligeorgiev at the University of Sofia in Bulgaria has developed a series of new intercalating dyes which have improved fluorescence staining characteristics for identification and analysis of DNA or RNA samples. These dyes have been demonstrated to have an increase in fluorescence of up to 1000 fold upon binding to DNA compared to the 10 fold increase seen with traditional ethidium bromide staining. The dyes are also highly stable allowing for prestaining of DNA with a very small amount of dye prior to running on a gel and then visualizing under appropriate wavelengths.

Marker Gene is working with Dr. Deligeorgiev to make these new dyes available for researchers here in the USA. To find out more about these reagents and methods, please see the references below or visit our website.

• Vasilev A, Deligeorgiev T, Kaloyanov S, Stoyanov S, Maximova V, Vaqueroc JJ, Alvarez-Buillac J (2010) "Synthesis of novel tetracationic asymmetric monomeric monomethine cyanine dyes - highly fluorescent dsDNA probes." Color. Technol., 127: 69-74.
• Kaloyanovaa S, Trusovab VM, Gorbenkob GP, Deligeorgieva T (2011) "Synthesis and fluorescence characteristics of novel asymmetric cyanine dyes for DNA detection." Journal of Photochemistry and Photobiology A: Chemistry 217: 147-156.
• Deligeorgieva TG, Kaloyanovaa S, Vaquero J (2009) "Intercalating Cyanine Dyes for Nucleic Acid Detection." Recent Patents on Materials Science 2: 1-26.
• Armitage BA, (2005) "Cyanine dye-DNA interactions: intercalation, groove binding, and aggregation." Top. Curr. Chem. 253: 55-76.
• Hinton DN, Bode VC,(1975) "Ethidium binding affinity of circular lambda deoxyribonucleic acid determined fluorometrically." J. Biol. Chem. 250 1061-1070.
• Richards GM,(1974) "Modifications of the diphenylamine reaction giving increased sensitivity and simplicity in the estimation of DNA", Anal. Biochem. 57
  369–376.
• Nafisi S, Saboury AA, Keramat N, Neault JF, Tajmir-Riahi HA,(2007) "Stability and structural features of DNA intercalation with ethidium bromide, acridine orange and methylene blue" J. Mol. Struct. 827: 35-43.
• Neidle S, Pearl LH, Herzyk P, Berman HM, (1988) "A molecular model for proflavine-DNA intercalation" Nucleic Acids Res. 16: 8999-9016.
• Daxhelet GA, Coene MM, Hoet P, Cocito CG,(1989) "Spectrofluorometry of dyes with DNAs of different base composition and conformation." Anal. Biochem. 179: 401-403.
• Deligeorgiev TG, Kaloyanova S, Vaquero JJ (2009) "Intercalating cyanine dyes for nucleic acid detection." Recent Pat. Mater. Sci. 2 1–26.
  
  
  

Loop-mediated Isothermal Amplification ( LAMP).

LAMP is a recently developed technique for quick and easy detection of a DNA or RNA sequences without the need for expensive equipment. This makes it an ideal tool for diagnosis of viruses and disease in samples from patients. LAMP operates on a similar model to PCR, with a similar level of sensitivity with the difference being LAMP is performed under isothermic conditions with results determined in 30-60 minutes. In order to detect the DNA or RNA in question, the reaction consists of a sample to be tested, which can contain as few as 7 copies of the target, 2-6 primers, an outer pair and 1 or 2 inner pairs, a strand disrupting polymerase, dNTPs, and if the sample is RNA, a reverse transcriptase. The reaction typically proceeds at 65^oC for up to 1 hour and is then stopped by incubation at 85^oC for 2 minutes. Positive reaction can be visualized either by change in turbidity of the reaction or by spectrophotometric quantitation. Alternatively, an intercalating dye can be added to the reaction with a positive result indicated by an increase in fluorescence.

Several practical applications of LAMP have been described including a test to distinguish Haemophilus influenzae from the genotypically similar Haemophilus parainfluenzae. Even more significantly, LAMP was able to detect HIV and has thus been used broadly for such detection in the field. Other applications have been described including determination of melon shelf life for its optimal breeding and for the detection of the Japanese Yam Mosiac Virus to prevent spreading of the virus in yam nurseries. For more information about these methods, please see the references below, or visit our website.

• Fang X, Li J, Chen Q (2008) "One New Method of Nucleic Acid Amplification—Loop-mediated Isothermal Amplification of DNA", Virologica Sinica 23(3):167-172.
• Torigoe H, Seki M, Yamashita Y, Sugaya A, Maeno M.(2007) "Detection of Haemophilus influenzae by loop-mediated isothermal amplification (LAMP) of the outer membrane protein P6 gene." Jpn. J. Infect. Dis. 60(1):55-8.
• Fukuta S, Mizukami Y, Ishida A, Kanbe M.(2006) "Development of loop-mediated isothermal amplification (LAMP)-based SNP markers for shelf-life in melon (Cucumis melo L.)." J. Appl. Genet. 47(4):303-8.
• Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T.(2000) "Loop-mediated isothermal amplification of DNA." Nucleic Acids Res. 28(12):E63
• Fukuta S, Iida T, Mizukami Y, Ishida A, Ueda J, Kanbe M, Ishimoto Y. (2003) "Detection of Japanese yam mosaic virus by RT-LAMP." Arch. Virol. 148(9):1713-1720.

Use of Intercalating Dyes for Super-Resolution Imaging of DNA.

With standard fluorescent microscopy the ability to visualize DNA is limited. Images are often blurred and histology difficult to analyze. However, by using fluorophores such as YOYO-1 or YO-PRO-1 that are photoswitchable, it can be possible to separate the emission of separate dyes and use this to precisely deduce their location. These dyes can undergo reversible photoswitching, where the fluorophore can be switched between a fluorescent state and a dark state intracellularly. By using cycles of switching and detection, the localization of single molecules on a wide-field microscopic view can be used to construct super-resolution images that show the localization density of the fluorophores with spatial resolution of approximately tens of nm.
With the advent of super-resolution microscopy and the use of these intercalating dyes, DNA topology can be seen at the nanoscale. These dyes are particularly useful as they bind to DNA non-covalently at a high density without sequence specificity and due to the high magnitude increase in fluorescence upon DNA binding there is little background interference from unbound dye. These dyes have been shown to photoblink under appropriate conditions of wavelength and buffer system and the photoblinking occurs at an appropriate rate to allow the creation of super-resolution images.
These techniques have shown several applications including tracking the movement of chromosomes during mitosis and examining the mechanisms and forces at work during their movement. The development of further dyes which bind densely to DNA will only further the applications of this level of imaging. For more information about these assays, please see the references below or visit our website.

• Flors C (2011) "DNA and chromatin imaging with super-resolution fluorescence microscopy based on single-molecule localization." Biopolymers 95: 290-297.
• Thomann D, Rines DR, Sorger PK, Danuser G (2002) "Automatic fluorescent tag detection in 3D with super-resolution: application to the analysis of chromosome movement". Journal of Microscopy 208: 49-64.
• Huang B, Bates M, Zhuang X (2009) "Super-resolution fluorescence microscopy" Annu. Rev. Biochem. 78:993-1016.
• Flors C (2010) "Photoswitching of monomeric and dimeric DNA-intercalating cyanine dyes for super-resolution microscopy applications" Photochemical & Photobiological Sciences 9:5 643-648.
  

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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).
• Confidentiality, help in patent preparation and filings.

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