A GUIDE TO NUCLEIC ACID LABELING AND DETECTION SYSTEMS VECTOR LABORATORIES S T A T E O F T H E A R T L A B E L I N G & D E T E C T I O N S Y S T E M S 1 Table of Contents Introduction ............................................................................................................................. 2-3 Labeling of Nucleic Acids ............................................................................................................ 4-9 Choosing a Labeling System Based on Nucleic Acid Type and Size.......................................................4 Choosing a Labeling System Based on Application and Label..............................................................5 PHOTOPROBE ® Reagents and the FastTag ® System.............................................................................6-7 5’ EndTag ™ and 3’ EndTag ™ Labeling Systems.....................................................................................8-9 Detection of the Nucleic Acid Label .......................................................................................................... 10-13 Antibody Conjugates..............................................................................................................................10 Complementary Detection Reagents...............................................................................................11-13 Fluorescent Systems................................................................................................................................11 VECTASHIELD ® Mounting Media...........................................................................................................12 Chromogenic Substrates for Enzyme-Based Detection........................................................................13 DuoLuX ™ Chemiluminescent/ Fluorescent Substrate..............................................................................13 Applications ................................................................................................................................................ 14-18 Southern and Northern Blot Hybridization..........................................................................................14 In Situ Hybridization...............................................................................................................................15 Optimizing Probe Length for In Situ Hybridization.............................................................................15 Comparative Genomic Hybridization....................................................................................................15 Cellular Localization...............................................................................................................................16 EndTag ™ - Labeled Primers Used in PCR Amplification..............................................................................16 Covalent Attachment of 5’ EndTag ™ -Labeled Nucleic Acids to Gold..................................................16 Affinity Binding......................................................................................................................................17 Resolve Same-Sized DNA in a Sequence-Specific Manner....................................................................18 Amplification of Fluorescent Signal in Nucleic Acid Microarrays........................................................18 Protocols ................................................................................................................................................. 19-26 Assessing Labeling Efficiency - Estimation of Labeling by Comparative Dot Blot..............................19 Assessing Labeling Efficiency - Quantitation of Biotin Using the Quant*Tag ™ Biotin Kit.................19 Southern Blot Hybridization and Detection....................................................................................20-21 FISH: Fluorescence Detection of Biotin-Labeled ISH Probes................................................................22 FISH: Fluorescence Detection of Fluorescein-Labeled ISH Probes.......................................................23 ISH: Chromogenic Detection of Biotin-Labeled ISH Probes................................................................24 ISH: Chromogenic Detection of Fluorescein-Labeled ISH Probes.......................................................25 Methods used to elute single strands of DNA from VECTREX ® - bound biotinylated DNA...........................26 Nucleic Acid Purification .............................................................................................................................27 Fast Facts ....................................................................................................................................................... 28 References .............................................................................................................................................. 29-31 Ordering Information .......................................................................................................................................... 32 Inside front cover : A plasmid biotinylated with the FastTag ® Biotin Labeling System was complexed with a lactosylated polyethylenimine and instilled intranasally to mice. On this tissue slide, the plasmid labeled with rhodamine-conjugated streptavidin (red) is detected in ep ithelial cells of the bronchus and in the pulmonary cells surrounding it. Image kindly supplied by G.Thévenot and Dr. I. Fajac, Faculté de Médecine Paris 5, Paris, France. 2 Introduction Detection of Nucleic Acid Labels Recognized as a leader in the development of Each choice of label is complemented by several de tection systems, Vector Laboratories has been different fluorescent or enzyme-based detection providing the research community with quality reagents providing the researcher with flexibility reagents yielding high sensitivity and low back - in optimizing experimental systems. Among the ground since 1976. Continuing in this tradition, ex tensive range of antibody conjugates and our molecular biology tools offer sensitivity, avidin/streptavidin conjugates, amplification relia bility, simplicity, and economy. These systems reagents such as Biotinylated Anti-Streptavidin and and reagents facilitate the labeling, detection, Biotinylated Anti-Avidin are also available. These and manipulation of nucleic acids. Microarrays, reagents reliably increase sensitivities of target blotting assays, cell trafficking, in situ hybridization, sig nals in microarray applications and fluorescence comparative genomic hybridization, subtractive in situ hybridization. In addition, the superior anti- hy bridization, and affinity purification are just a fading property of the VECTASHIELD ® line of few applications in which our molecular biology mounting media ensures the preservation of signal tools can be successfully employed. intensity in applications such as fluorescence in situ hybridization. (Please see pages 10-13 for a listing Labeling of Nucleic Acids of selected products.) The PHOTOPROBE ® reagents, FastTag ® Systems, For nucleic acid blotting applications, the Vector ® and the 3’ and 5’ EndTag ™ Labeling Systems are UltraSNAP ™ Detection System (page 14) containing efficient and reliable labeling methods that specially formulated reagents detects biotinylated offer choices for optimum labeling for a given probes with high sensitivity and low background. ap plica tion, as well as choices for incorporating This system utilizes the DuoLuX ™ Chemiluminescent/ fluorochromes, haptens, or affinity tags. Fluorescent Substrate with in tense and prolonged light emission characteristics, ideal for Southern and Nucleic acid probes for applications such as in situ northern hybridization, dot blot, plaque or colony hybridization, northern and Southern blot screening. hy bridization, comparative genomic hybridization, intracellular localization experiments, and DNA microarrays can be easily and optimally labeled and subsequently visualized with our comprehen - sive range of fluorescent, chemiluminescent, or chromogenic detection systems. FastTag ® Fluorescein labeled pUC1.77 (yellow-green) detected Cellular uptake of labeled plasmid: FastTag ® Fluorescein labeled with Biotinylated Anti-Fluorescein and Fluorescein Avidin DCS. plasmid DNA (green) incubated with COS-7 cells. Nuclei were Nuclei were counterstained and mounted with VECTASHIELD ® counterstained and mounted with VECTASHIELD ® with DAPI with PI (red-orange). (blue). www.vectorlabs.com 3 Immobilization of Labeled Probes Quantitation of Biotin using the Quant *Tag ™ System Vector Laboratories offers both irreversible The Quant*Tag ™ Biotin Kit (page 19) is designed to (VEC TREX ® Avidin D) as well as reversible determine the amount of free biotin in solution or (VECTREX ® Avidin DLA or VECTREX ® AAL) affinity the number of biotins attached to proteins, nucleic binding matrices. Applications like genomic/ cDNA acids or other macromolecules. Samples do not subtraction or library screening by hybrid capture need to be predigested. The kit reagents chemically require labeled nucleic acids to be immobilized react with free or bound biotin, producing a colored onto a solid support. Using our affinity binding product that can be quantified using a spectropho - matrices in such applications can yield information tometer. The absorbance is measured in the visible about differential gene expression or allow the spectrum, allowing the