chapter 8—nucleic acid detection and analysis

CHAPTER 8

Nucleic Acid Detection and Analysis

Molecular Probes HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probes Resources

Molecular Probes Handbook (online version)Comprehensive guide to uorescent probes and labeling technologies

lifetechnologies.com/handbook

Fluorescence SpectraViewerIdentify compatible sets of uorescent dyes and cell structure probes

lifetechnologies.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

lifetechnologies.com/bioprobes

Access all Molecular Probes educational resources at lifetechnologies.com/mpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi sher.com/handbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi sher.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi sher.com/bioprobes

Access all Molecular Probes educational resources at thermofi sher.com/probes

http://thermofisher.com/handbookhttp://thermofisher.com/spectraviewerhttp://thermofisher.com/bioprobeshttp://thermofisher.com/probes

303www.invitrogen.com/probes

The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

EIG

HT

CHAPTER 8

Nucleic Acid Detection and Analysis

8.1 Nucleic Acid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307Properties of Cyanine Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Premier Cyanine Dyes for Ultrasensitive Nucleic Acid Detection and Quantitation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307

Cell-Impermeant Cyanine Dimers: The TOTO Family of Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

High Anity for Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308

High Fluorescence Enhancements and High Quantum Yields upon Binding to Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Modifying the Dimers Creates Compounds with Dierent Spectral Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Binding Modes of the Cyanine Dimers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309

Working with Cyanine Dimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

Cell-Impermeant Cyanine Monomers: The TO-PRO Family of Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

Spectral Characteristics of the Cyanine Dye Monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310

Working with Cyanine Monomers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

Cell-Impermeant SYTOX Dyes for Dead-Cell Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

SYTOX Green Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

SYTOX Blue Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311

SYTOX Orange Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

Cell-Permeant Cyanine Dyes: The SYTO Nucleic Acid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

SYTO Nucleic Acid Stains for DNA and RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

SYTO RNASelect Green-Fluorescent Cell Stain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

Amine-Reactive Cyanine Dye. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314

Phenanthridines and Acridines: Classic Intercalating Dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

Cell-Impermeant Ethidium Bromide and Propidium Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

Cell-Permeant Hexidium Iodide. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

Cell-Permeant Dihydroethidium (Hydroethidine) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

High-Anity Ethidium Homodimers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317

Ethidium Monoazide: A Photocrosslinking Reagent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

Acridine Orange: A Dual-Fluorescence Nucleic Acid Stain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

AT-Selective Acridine Homodimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 318

AT-Selective ACMA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Indoles and Imidazoles: Classic Minor GrooveBinding Dyes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

DNA-Selective Hoechst Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

AT-Selective DAPI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Other Nucleic Acid Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

7-Aminoactinomycin D and Actinomycin D: Fluorescent Intercalators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Hydroxystilbamidine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Long-Wavelength LDS 751 Dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

NeuroTrace Fluorescent Nissl Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321

Data Table 8.1 Nucleic Acid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

Product List 8.1 Nucleic Acid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

thermofi sher.com/probes



Chapter 8 Nucleic Acid Detection and Analysis

8.2 Labeling Oligonucleotides and Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326ChromaTide Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

Structures of the ChromaTide Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Fluorescent ChromaTide Nucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Using ChromaTide Nucleotides in Enzymatic Labeling Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Amine-Modied Nucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Unlabeled and Labeled aha-dUTP and aha-dCTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

Aminoallyl dUTP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Alexa Fluor Amine-Reactive Dye Decapacks for Labeling Amine-Modied DNA and RNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

ARES DNA Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

FISH Tag DNA and FISH Tag RNA Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332

ULYSIS Nucleic Acid Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333

Labeled Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

Panomer 9 Random Oligodeoxynucleotides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

Alexa Fluor Oligonucleotide Amine Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334

Labeling Amine-, Thiol- or Phosphate-Modied Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Amine-Reactive SYBR Dye . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Dyes for Sequencing Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335

Other Labeling Methods for Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Labeling Nascent DNA and RNA for Cell Proliferation Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Labeling Abasic Sites with ARP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

Labeling Cytidine Residues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

Specialized Methods for Nucleic Acid Modication. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

Product List 8.2 Labeling Oligonucleotides and Nucleic Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

8.3 Nucleic Acid Quantitation in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339Quant-iT Assay Kits for DNA and RNA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339

Quant-iT DNA Assay Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340

Quant-iT RNA Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Quant-iT Assay Kits for Use with the Qubit Fluorometer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341

Quant-iT PicoGreen dsDNA Quantitation Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342

Quant-iT OliGreen ssDNA Quantitation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343

Quant-iT RiboGreen RNA Quantitation Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

Quant-iT RiboGreen RNA Reagent and Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344

RediPlate 96 RiboGreen RNA Quantitation Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 345

Other Stains for Nucleic Acid Quantitation in Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Cyanine Dyes and Phenanthridine Dyes for Nucleic Acid Quantitation in Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Hoechst 33258 Dye for Quantitating DNA in Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346

Real-Time Quantitative PCR Using SYBR Green I Nucleic Acid Gel Stain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

Product List 8.3 Nucleic Acid Quantitation in Solution. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




8.4 Nucleic Acid Detection on Gels, Blots and Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349Nucleic Acid Detection in Gels. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

SYBR Gold Nucleic Acid Gel Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349

SYBR Green I Nucleic Acid Gel Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 350

SYBR Green II RNA Gel Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

SYBR Green Nucleic Acid Gel Stains: Special Packaging and a Starter Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

SYBR Safe DNA Gel Stain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

Ethidium Bromide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Cyanine Monomers for Staining DNA in Electrophoretic Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Cyanine and Ethidium Dimers for Staining DNA Prior to Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Electrophoretic Mobility-Shift (Bandshift) Assay (EMSA) Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

Other Nucleic Acid Stains for Gel-Staining Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

Safe Imager Blue-Light Transilluminator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

SYBR Photographic Filters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356

SYBR Photographic Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

SYBR Safe Photographic Filter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Capillary Electrophoresis and Channel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Capillary Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Channel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

Quality Control Testing on Microarrays and Blots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

Nucleic Acid Stains for Standardizing Microarrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Panomer Random-Sequence Oligonucleotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

Product List 8.4 Nucleic Acid Detection on Gels, Blots and Arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360



thermofisher.com/probes




TO-PRO-3 iodide, MitoTracker Orange CMTMRos and BODIPY FL phallacidin.






Section 8.1 Nucleic Acid Stains

We oer an extensive assortment of nucleic acid stains, many of which have been developed in our research laboratories. is section discusses the physical properties of the various classes of dyes listed below. e sections that follow describe applications of these nucleic acid stains for genom-ics research. e four classes of Molecular Probes cyanine dyes include:

Premier dyes for ultrasensitive nucleic acid quantitation and gel staining (Table 8.1)

Cell-impermeant TOTO, TO-PRO and SYTOX families of dyes (Table 8.2)

Cell-permeant SYTO family of dyes (Table 8.3) Amine-reactive SYBR dye that can be used to form bioconjugates

e three classes of classic nucleic acid stains (Table 8.4) include:

Intercalating dyes (Figure 8.1.1), such as ethidium bromide and propidium iodide

Minor-groove binders (Figure 8.1.1), such as DAPI and the Hoechst dyes

Other types of nucleic acid binders (Figure 8.1.1) such as acridine orange, 7-AAD, LDS 751 and hydroxystilbamidine

Properties of Cyanine DyesMolecular Probes nucleic acidbinding cyanine dyes share several

important spectroscopic and physical properties:

High molar absorptivity, with extinction coecients typically greater than 50,000 cm1M1 at visible wavelengths

Very low intrinsic uorescence, with quantum yields usually less than 0.01 when not bound to nucleic acids

Large uorescence enhancements (oen over 1000-fold) upon bind-ing to nucleic acids, with increases in quantum yields to as high as 0.9

Moderate to very high anity for nucleic acids, with little or no staining of other biopolymers

eir uorescence aborption and emission spectra span the visible-light spectrum from blue to near-infrared (Figure 8.1.2) with additional absorption peaks in the UV, making them compatible with many dif-ferent types of instrumentation. e cyanine dyes also show important dierences in some physical characteristicsparticularly in cell mem-brane permeability and nucleic acid specicitythat allow their dis-tribution into distinct classes, and these classes are discussed in detail below and in the following sections of this chapter.

Premier Cyanine Dyes for Ultrasensitive Nucleic Acid Detection and Quantitation

Several of our cyanine dyes provide excellent sensitivity in specic nucleic acid assays (Table 8.1). For these dyes, we have developed de-tailed and extensively tested protocols to facilitate reproducible, high-sensitivity results in these assays.

8.1 Nucleic Acid Stains

Figure 8.1.1 Schematic diagram showing the dierent binding modes of dyes (and other ligands) to DNA.

Intercalator

Major groove binder

External binder

Minor groove binder

Bis-intercalator

Major groove

Minor groove

Figure 8.1.2 Normalized uorescence emission spectra of DNA-bound cyanine dimers, iden-tied by the color key on the sidebar.

400 500 600 700 800

Fluo

resc

ence

em

issi

on

Wavelength (nm)

POPO-1BOBO-1YOYO-1TOTO-1JOJO-1POPO-3LOLO-1BOBO-3YOYO-3TOTO-3

PicoGreen, OliGreen and RiboGreen quantitation reagents and their Quant-iT reagent counterparts in Section 8.3 set a bench-mark for the detection and quantitation of DNA, oligonucleotides and RNA in solution. ese reagents oer extremely simple and rapid protocols as well as linear ranges that span up to four orders of magnitude in nucleic acid concentration.

SYBR Gold, SYBR Green I and SYBR Green II nucleic acid gel stains in Section 8.4 are ultrasensitive gel stains that surpass the sensitivity of ethidium bromide by more than an order of magni-tude in nucleic acid detection.

SYBR Safe DNA gel stain (Section 8.4) is not only signicantly less mutagenic than ethidium bromide, but SYBR Safe stains detection sensitivity is comparable to that of ethidium bromide. SYBR Safe stain showed no or very low mutagenic activity when tested by an independent, licensed testing laboratory, and it is not classied as hazardous waste under U.S. Federal regulations.

CyQUANT GR dye (C7026), discussed in Section 15.4, is a cyanine dye designed to quantitate cell proliferation and can reliably detect the nucleic acids in as few as 50 cells.








Cell-Impermeant Cyanine Dimers: The TOTO Family of Dyes

e cyanine dimer dyes listed in Table 8.2sometimes referred to as the TOTO family of dyesare symmetric dimers of cyanine dyes with exceptional sensitivity for nucleic acids. is sensitivity is due to a high anity for nucleic acids, in combination with a very high uo-rescence enhancement and quantum yield upon binding. e unique physical characteristics of these dyes and some illustrative applications are discussed below; specic applications are discussed in later sections of this chapter.

Each of the cyanine dimer dyes is available separately. For research-ers designing new applications, the Nucleic Acid Stains Dimer Sampler Kit (N7565) provides samples of eight spectrally distinct analogs of the dimeric cyanine dyes for testing (Table 8.2).

High Anity for Nucleic AcidsAppropriately designed dimers of nucleic acidbinding dyes have

nucleic acidbinding anities that are several orders of magnitude greater than those of their parent monomer dyes.13 For example, the in-trinsic DNA binding anity constants of ethidium bromide and ethid-ium homodimer-1 (E1169) are reported to be 1.5 105 and 2 108 M1, respectively, in 0.2 M Na+.4 As a result, the dimeric cyanine dyes are among the highest-anity uorescent probes available for nucleic acid staining.

For example, in the TOTO-1 dimeric cyanine dye (T3600), the positively charged side chains of the TO-PRO-1 monomeric cyanine dye (T3602, Figure 8.1.3) are covalently linked to form the TOTO-1 molecule, with four positive charges (Figure 8.1.4). is linkage gives TOTO-1 dye a greatly enhanced anity for nucleic acidsmore than 100 times greater than that of the TO-PRO-1 monomer. TOTO-1 dye

Table 8.2 Cell membraneimpermeant cyanine nucleic acid stains.

Cat. No. Dye Ex/Em*

SYTOX Dyes: Dead-Cell StainsS11348S34857

SYTOX Blue 445/470

S7020 SYTOX Green 504/523S11368 SYTOX Orange 547/570S34859 SYTOX Red 640/658

Cyanine Dimers: High-Anity StainsP3580 POPO-1 434/456B3582 BOBO-1 462/481Y3601 YOYO-1 491/509T3600 TOTO-1 514/533J11372 JOJO-1 529/545P3584 POPO-3 534/570L11376 LOLO-1 565/579B3586 BOBO-3 570/602Y3606 YOYO-3 612/631T3604 TOTO-3 642/660N7565 Dimer Sampler Kit Various

Cyanine Monomers: Nuclear Counterstains**P3581 PO-PRO-1 435/455Y3603 YO-PRO-1 491/509T3602 TO-PRO-1 515/531J11373 JO-PRO-1 530/546P3585 PO-PRO-3 539/567Y3607 YO-PRO-3 612/631T3605 TO-PRO-3 642/661T7596 TO-PRO-5 747/770*Wavelengths of excitation (Ex) and emission (Em) maxima, in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution. Products supplied as 250 L of a 5 mM solution. Products (except N7565) supplied as 200 L of a 1 mM solution. Includes 10 L each of a 1 mM solution of the TOTO-1, TOTO-3, YOYO-1, YOYO-3, BOBO-1, BOBO-3, POPO-1 and POPO-3 dyes. **Products supplied as 1 mL of a 1 mM solution.

Figure 8.1.3 TO-PRO-1 iodide (515/531, T3602). Figure 8.1.4 TOTO-1 iodide (514/533, T3600).

Table 8.1 Specialty nucleic acid reagents for molecular biology.

Cat. No. Dye Ex/Em* Application

Dyes for Ultrasensitive Solution Quantitation

P7581, P11495, P7589, P11496 Quant-iT PicoGreen dsDNA reagent and Kits 502/523 Ultrasensitive reagent for solution quantitation of dsDNA

O7582, O11492 Quant-iT OliGreen ssDNA reagent and Kit 498/518 Ultrasensitive reagent for solution quantitation of ssDNA and oligonucleotides

R11491, R11490, R32700 Quant-iT RiboGreen RNA reagent and Kits 500/525 Ultrasensitive reagent for solution quantitation of RNA

Dyes for Sensitive Detection of Nucleic Acids in Gels and on Blots

S11494 SYBR Gold nucleic acid gel stain 495/537 Ultrasensitive gel stain for single- or double-stranded DNA or RNA post-electrophoresis

S7563, S7567, S7585 SYBR Green I nucleic acid gel stain 494/521 Ultrasensitive gel stain for double-stranded DNA and oligonucleotides post-electrophoresis; also useful for real-time PCR assays

S7564, S7568, S7586 SYBR Green II RNA gel stain 492/513 Sensitive stain for RNA and single-stranded DNA post-electrophoresis

S33100, S33101, S33102, S33110, S33111, S33112

SYBR Safe DNA stain 502/530 Sensitive DNA gel stain with signicantly reduced mutagenicity

* All excitation (Ex) and emission (Em) maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution. In addition to these dyes, we oer a wide selection of Quant-iT Kits for use with the Qubit uorometer and with microplate readers; these are listed in Table 8.8.







exhibits a higher anity for double-stranded DNA (dsDNA) than even the ethidium homodi-mers and also binds to both single-stranded DNA (ssDNA) and RNA. e extraordinary sta-bility of TOTO-1nucleic acid complexes 2,5,6 allows the dyeDNA association remains stable, even during electrophoresis (Figure 8.1.5); thus, samples can be stained with nanomolar dye concentrations prior to electrophoresis,7,8 thereby reducing the hazards inherent in handling large volumes of ethidium bromide staining solutions.2,6,9 In contrast, the binding of thiazole orangethe parent compound of TOTO-1 and TO-PRO-1is rapidly reversible, limiting the dyes sensitivity and rendering its nucleic acid complex unstable to electrophoresis.9

High Fluorescence Enhancements and High Quantum Yields upon Binding to Nucleic Acids

In addition to their superior binding properties, TOTO-1 dye and the other cyanine dimers are essentially nonuorescent in the absence of nucleic acids and exhibit uorescence enhance-ments upon DNA binding of 100- to 1000-fold,5,10 which compares favorably with the uores-cence enhancement of thiazole orange upon DNA binding 11 (~3000-fold). Furthermore, the uorescence quantum yields of the cyanine dimers bound to DNA are high (generally between 0.2 and 0.6), and their extinction coecients are an order of magnitude greater than those of the ethidium homodimers.5 is sensitivity is sucient for detecting single molecules of labeled nucleic acids by optical imaging (Figure 8.1.6) and ow cytometry and for tracking dye-labeled virus particles in microbial communities and aquatic systems by uorescence microscopy.12,13 ese dyes are generally considered to be cell impermeant, although their use to stain reticulo-cytes permeabilized by 5% DMSO has been reported.14

Modifying the Dimers Creates Compounds with Dierent Spectral Characteristics

Simply by changing the aromatic rings and the number of carbon atoms linking the cyanine monomers, we were able to synthesize an extended series of these dyes with dierent spectral characteristics (Table 8.2). Chemical modications produce dramatic shis in the absorption and emission spectra and reduce the quantum yields of the bound dyes but cause little or no change in their high anity for DNA. e names of the dyes reect their basic structure and spectral characteristics. For example, YOYO-1 iodide (491/509) has one carbon atom bridg-ing the aromatic rings of the oxacyanine dye and exhibits absorption/emission maxima of 491/509 nm when bound to dsDNA. YOYO-3 dye (612/631)which diers from YOYO-1 dye only in the number of bridging carbon atomshas absorption/emission maxima of 612/631 nm when bound to dsDNA. Fluorescence spectra for the POPO, BOBO, YOYO, TOTO, JOJO and LOLO dyes bound to dsDNA are shown in Figure 8.1.2. e spectra of these dyes at dye:base ratios of less than 1:1 are essentially the same for the corresponding dyessDNA and dyeRNA complexes. At higher dye:base ratios, however, ssDNA and RNA complexes of all of the mono-methine ("-1") dyes of the TOTO series and TO-PRO series have red-shied emissions, whereas corresponding complexes of the trimethine ("3") analogs do not. us, the cyanine dimer family provides dyes with a broad range of spectral characteristics to match the output of almost any available excitation source.

Binding Modes of the Cyanine Dimerse studies on cyanine dimer binding modes have focused on the YOYO-1 and TOTO-1

dyes. YOYO-1 dye was found to exhibit at least two distinct binding modes. At low dye:base pair ratios, the binding mode appears to consist primarily of bis-intercalation 1519 (Figure 8.1.1). Each monomer unit intercalates between bases, with the benzazolium ring system sandwiched be-tween the pyrimidines and the quinolinium ring between the purine rings, causing the helix to unwind. e distortion in the local DNA structure caused by YOYO-1 bis-intercalation has been observed by two-dimensional NMR spectroscopy. At high dye:base pair ratios, a second, less well characterized mode of external binding begins to contribute.15,16 Circular dichroism measurements also indicate a possible dierence in the binding modes of YOYO-1 dye to ss-DNA and dsDNA. ese data are consistent with our own results, including the observation that the uorescence emission of the YOYO-1 dye complex with nucleic acids shis to longer wavelengths at high dye:base ratios upon binding to single-stranded nucleic acids and that the salt, ethanol and sodium dodecyl sulfate (SDS) sensitivity of YOYO-1 dye binding to DNA is a function of the dye:base pair ratio.19

Figure 8.1.5 Lambda bacteriophage HindIII fragments were prestained with various nucleic acid dyes, run on a 0.7% agarose gel and visualized using a standard 300 nm UV transilluminator. From left to right, the dyes used were: POPO-1 (P3580), BOBO-1 (B3582), YOYO-1 (Y3601), TOTO-1 (T3600), JOJO-1 (J11372), POPO-3 (P3584), LOLO-1 (L11376), BOBO-3 (B3586), YOYO-3 (Y3606) and TOTO-3 (T3604) nucleic acid stains. The longest-wave-length stains are barely visible to the eye but can be detect-ed with infrared-enhanced lms and imaging equipment.

Figure 8.1.6 The relaxation of a single, 39 mlong DNA mol-ecule stained with YOYO-1 iodide (Y3601) imaged at 4.5 sec-ond intervals. After the 1 m polystyrene sphere was trapped with optical tweezers, the attached DNA was stretched to its full extension in a uid ow and then allowed to relax upon stoppage of uid ow due to its entropic elasticity (Science (1994) 264:822). The YOYO-1 iodideDNA complex is ex-cited with the 488 nm spectral line of the argon-ion laser and visualized through a 515 nm longpass optical lter using a Hamamatsu SIT camera with image processing. Image con-tributed by Thomas Perkins, Stanford University.








TOTO-1 dye is also capable of bis-intercalation,20 although it reportedly interacts with ds-DNA and ssDNA with similarly high anity.1 NMR studies of TOTO-1 dye interactions with a double-stranded 8-mer indicate that TOTO-1 dye is a bis-intercalator, with the uorophores intercalating between the bases and the linker region having interactions in the minor groove 21 (Figure 8.1.7). Binding of the dye partially unwinds the DNA,21 distorting and elongating the helix.22 However, another study using uorescence polarization measurements suggests that an external binding mode, where the dipole of the dye molecule is aligned with the DNA grooves, may be more important.23 TOTO-1 dye reportedly exhibits some sequence selectivity for the site 5-CTAG-3 , although it will bind to almost any sequence in dsDNA.2427 TOTO-1 dye does not exhibit cooperative binding to DNA, suggesting that it may be a suitable dye for detecting nucleic acids in gels.25

e binding modes of the other members of the TOTO dye series have also been partially characterized. Electrophoresis and uorescence lifetime measurements have shown that YOYO-3 dye also appears to intercalate into DNA.28 During application development, we have deter-mined that staining of nucleic acids by BOBO-1 and POPO-1 dyes is much faster (occurring within minutes) than staining by YOYO-1 or TOTO-1 dyes (which can take several hours to reach equilibrium under the same experimental conditions),20 indicating possible dierences in their binding mechanisms. Fluorescence yield and lifetime measurements have been used to as-sess the base selectivity of an extensive series of these dyes.10 Circular dichroism measurements have shown that bis-intercalation is the predominant binding mode for the POPO-1 dye.29

Working with Cyanine DimersAll of the dyes in the TOTO series (Table 8.2) are supplied as 1 mM solutions in dimethyl-

sulfoxide (DMSO), except for POPO-3 (P3584), which is supplied as a 1 mM solution in dimeth-ylformamide (DMF). ese cationic dyes appear to be readily adsorbed out of aqueous solutions onto surfaces (particularly glass) but are very stable once complexed to nucleic acids.

Cell-Impermeant Cyanine Monomers: The TO-PRO Family of Dyes

Our TO-PRO family of dyes, all of which are listed in Table 8.2, each comprise a single cyanine dye and a cationic side chain (Figure 8.1.3). e monomeric dyes in the TO-PRO series are spectrally analogous to the corresponding dimeric cyanine dyes; however, with only two positive charges and one intercalating unit, the TO-PRO dyes exhibit somewhat reduced anity for nucleic acids relative to the dyes in the TOTO series. Like their dimeric counterparts, these monomeric cyanine dyes are typically impermeant to cells,30 although YO-PRO-1 (Y3603) dye has been shown to be permeant to apoptotic cells, providing a convenient indicator of apopto-sis 3134 (Section 15.5). YO-PRO-1 has also been observed to pass through P2X7 receptor channels of live cells.3537

Spectral Characteristics of the Cyanine Dye Monomerse TO-PRO family of dyes retains all of the exceptional spectral properties of the di-

meric cyanine dyes discussed above. e absorption and emission spectra of these monomeric cyanine dyes cover the visible and near-infrared spectrum (Table 8.2). ey also have rela-tively narrow emission bandwidths, thus facilitating multicolor applications in imaging and ow cytometry. YO-PRO-1 (491/509) and TO-PRO-1 (515/531) dyes are optimally excited by the 488 nm and 514 nm spectral lines of the argon-ion laser, respectively. In ow cytometric analysis, the TO-PRO-3 (642/661) complex with nucleic acids has been excited directly by the red He-Ne laser 38 and indirectly by the argon-ion laser by using uorescence resonance energy transfer (FRET) from co-bound propidium iodide 39 (Fluorescence Resonance Energy Transfer (FRET)Note 1.2). e TO-PRO-3 complex with nucleic acids has also been de-tected in a ow cytometer equipped with an inexpensive 3 mW visible-wavelength diode laser that provides excitation at 635 nm.40 Although the DNA-induced uorescence enhancement of TO-PRO-5 dye (T7596) is not as large as that observed with our other cyanine dyes, its spec-tral characteristics (excitation/emission maxima ~745/770 nm) provide a unique alternative for multicolor applications.

Figure 8.1.7 NMR solution structure of the TOTO-1 dye (T3600) bound to DNA; the image was derived from data submitted to the Protein Data Bank (number PDB 108D, www.rcsb.org/pdb/) (Nucleic Acids Res (2000) 28:235). The NMR structure shows that TOTO-1 binds to DNA through bis-intercalation (Biochemistry (1995) 34:8542).

Figure 8.1.8 Absorption and uorescence emission spectra of the SYTOX Green nucleic acid stain bound to DNA.







Working with Cyanine Monomerse binding anity of the TO-PRO series of dyes to dsDNA is lower than that of the TOTO

series of dyes but is still very high, with dissociation constants in the micromolar range.41 TO-PRO dyes also bind to RNA and ssDNA, although typically with somewhat lower uorescence quantum yields. Fluorescence polarization studies indicate that TO-PRO-1 and PO-PRO-1 dyes bind by intercalation, with unwinding angles of 2 and 31, respectively.29 Binding of these dyes to dsDNA is not sequence selective.42 All dyes of the TO-PRO series (Table 8.2) are supplied as 1 mM solutions in DMSO.

Cell-Impermeant SYTOX Dyes for Dead-Cell StainingSYTOX Green Stain

Our SYTOX nucleic acid stains (Table 8.2) are cell-impermeant cyanine dyes that are par-ticularly useful as dead-cell stains. SYTOX Green nucleic acid stain (S7020) is a high-anity nucleic acid stain that easily penetrates cells with compromised plasma membranes and yet will not cross the membranes of live cells. It is especially useful for staining both gram-positive and gram-negative bacteria, in which an exceptionally bright signal is required. Following brief incubation with the SYTOX Green stain, dead cells uoresce bright green when excited with the 488 nm spectral line of the argon-ion laser or with any other 450500 nm source (Figure 8.1.8). Because all of the SYTOX dyes are essentially nonuorescent in aqueous medium, no wash steps are required. Unlike the DAPI or Hoechst dyes, the SYTOX Green nucleic acid stain shows little base selectivity. ese properties, combined with its ~1000-fold uorescence enhancement upon nucleic acid binding and high quantum yield, make our SYTOX Green stain a simple and quantitative single-step dead-cell indicator for use with epiuorescence and confocal laser-scanning microscopes, uorometers, uorescence microplate readers and ow cytometers (Figure 8.1.9).

e SYTOX Green nucleic acid stain can be used with blue- and red-uorescent labels for multiparameter analyses (Figure 8.1.10). It is also possible to combine the SYTOX Green nucleic acid stain with the SYTO 17 red-uorescent nucleic acid stain (S7579) for two-color visualization of dead and live cells. Because the SYTOX Green nucleic acid stain is an excellent DNA counterstain for chromosome labeling and for xed cells and tissues (Figure 8.1.11), we have incorporated it into our SelectFX Nuclear Labeling Kit (S33025), which is discussed in Section 12.5.

SYTOX Blue StainSYTOX Blue stain (S11348, S34857) is a high-anity nucleic acid stain that typically pen-

etrates only cells with compromised plasma membranes (Figure 8.1.12). e SYTOX Blue stain

Figure 8.1.11 Adult zebrash gut cryosections that have been incubated with BODIPY TR-X phallacidin (B7464), fol-lowed by the SYTOX Green nucleic acid stain (S7020), and then dehydrated and mounted. The image was obtained by taking multiple exposures through bandpass optical lter sets appropriate for uorescein and the Texas Red dye.

Figure 8.1.12 A mixed population of live and isopropyl alcoholkilled Micrococcus luteus stained with SYTOX Blue nucleic acid stain (S11348), which does not penetrate intact plasma membranes. Dead cells exhibit bright blue-uores-cent staining. The image was acquired using a longpass op-tical lter set appropriate for the Cascade Blue dye.

Figure 8.1.9 Quantitative ow cytometric analysis of Escherichia coli viability using the SYTOX Green nucleic acid stain (S7020). A bacterial suspension containing an equal number of live and isopropyl alcoholkilled E. coli was stained with SYTOX Green and analyzed using excitation at 488 nm. A bivariate frequency distribution for forward light scatter versus log uorescence intensity (collected with a 510 nm longpass optical lter) shows two clearly dis-tinct populations. When live and dead bacteria were mixed in varying proportions, a linear relationship between the population numbers and the actual percentage of live cells in the sample was obtained (see inset).

Figure 8.1.10 Bovine pulmonary artery endothelial cells (BPAEC) incubated with the xable, mitochondrion-selective MitoTracker Red CMXRos (M7512). After staining, the cells were formaldehyde-xed, acetone-permeabilized, treated with DNase-free RNase and counterstained using SYTOX Green nucleic acid stain (S7020). Microtubules were labeled with a mouse monoclonal anti-tubulin antibody, biotin-XX goat antimouse IgG antibody (B2763) and Cascade Blue NeutrAvidin biotin-binding protein (A2663). This photograph was taken using multiple exposures through bandpass optical lters appropriate for Texas Red dye, uorescein and DAPI using a Nikon Labophot 2 microscope equipped with a Quaduor epi-illumination system.








labels both DNA and RNA with extremely bright uorescence centered near 470 nm (Figure 8.1.13). e absorption maximum of the nucleic acidbound SYTOX Blue stain (~445 nm) permits very ecient uorescence excitation by the 436 nm spectral line of the mercury-arc lamp. Unlike many blue-uorescent dyes, the SYTOX Blue stain is also eciently excited by tungstenhalogen lamps and other sources that have relatively poor emission in the UV por-tion of the spectrum. e brightness of the SYTOX Blue stain allows sensitive detection with uorometers, microplate readers, arc-lampequipped ow cytometers and epiuorescence microscopes, including those not equipped with UV-pass optics.

Figure 8.1.14 Absorption and uorescence emission spec-tra of SYTOX Orange nucleic acid stain bound to DNA.

Table 8.3 Cell-permeant cyanine nucleic acid stains.

Cat. No. Dye* Ex/Em

Blue-Fluorescent SYTO dyes

S11351 SYTO 40 blue-uorescent nucleic acid stain 419/445




S11350 SYTO Blue-Fluorescent Nucleic Acid Stain Sampler Kit (SYTO dyes 4045)

Various

Green-Fluorescent SYTO Dyes

S32703 SYTO RNASelect green-uorescent cell stain 490/530

S34854 SYTO 9 green-uorescent nucleic acid stain 483/503


S34855 SYTO BC green-uorescent nucleic acid stain 485/500


S7578 SYTO 16 green-uorescent nucleic acid stain** 488/518







S7572 SYTO Green-Fluorescent Nucleic Acid Stain Sampler Kit (SYTO dyes 1114, 16, 21, 24 and 25)

Various

Orange-Fluorescent SYTO dyes

S11362 SYTO 81 orange-uorescent nucleic acid stain 530/544






S11360 SYTO Orange-Fluorescent Nucleic Acid Stain Sampler Kit (SYTO dyes 8085)

Various

Red-Fluorescent SYTO dyes

S11346 SYTO 64 red-uorescent nucleic acid stain 598/620







S11340 SYTO Red-Fluorescent Nucleic Acid Stain Sampler Kit (SYTO dyes 17, 5964)

Various

*All products supplied as 250 L of a 5 mM solution, with exceptions noted. Wavelengths of excitation (Ex) and emission (Em) maxima, in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution, except for the SYTO RNASelect green-uorescent cell stain, which was determined for the dye bound to Escherichia coli RNA. Supplied as individual 50 L vials. Unit size = 100 L. **Supplied as 250 L of a 1 mM solution.

Figure 8.1.13 Absorption and uorescence emission spec-tra of SYTOX Blue nucleic acid stain bound to DNA.







Figure 8.1.15 Human neutrophil nuclei stained with SYTO 13 live-cell nucleic acid stain (S7575). The photo was ac-quired using an optical lter appropriate for uorescein, and dierential interference contrast (DIC) sequentially in a Nikon Eclipse E800 microscope.

In a side-by-side comparison with the SYTOX Green stain, the SYTOX Blue stain yield-ed identical results when quantitating membrane-compromised bacterial cells. Furthermore, like the SYTOX Green stain, the SYTOX Blue stain does not interfere with bacterial cell growth. Because their emission spectra overlap somewhat, we have found that it is not ideal to use the SYTOX Blue stain and green-uorescent dyes together; however, uorescence emission of the SYTOX Blue stain permits clear discrimination from orange- or red-uorescent probes, facilitating the development of multicolor assays with minimal spectral overlap between signals.

SYTOX Orange StainSYTOX Orange nucleic acid stain (S11368) is designed to clearly distinguish dead bacteria,

yeast or mammalian cells from live cells. As compared with propidium iodide, SYTOX Orange stain has shorter-wavelength emission and its spectra more closely matches the rhodamine lter set (Figure 8.1.14). In addition, the SYTOX Orange stain has a much higher molar absorptivity (extinction coecient) than propidium iodide and a far greater uorescence enhancement upon binding DNA, suggesting that it may have a higher sensitivity as a dead-cell stain or as a nuclear counterstain. e SYTOX Orange stain was shown to be extremely useful for DNA fragment sizing by single-molecule ow cytometry when using a Nd:YAG excitation source, with a 450-fold enhancement upon binding to dsDNA.43

Cell-Permeant Cyanine Dyes: The SYTO Nucleic Acid StainsSYTO Nucleic Acid Stains for DNA and RNA

e SYTO dyes are somewhat lower-anity nucleic acid stains that passively diuse through the membranes of most cells. ese UV- or visible lightexcitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in gram-positive and gram-negative bacteria. We have synthesized a large number of SYTO dyes (Table 8.3) that share several important characteristics:

Permeability to virtually all cell membranes, including mammalian cells and bacteria (Chapter 15)

High molar absorptivity, with extinction coecients greater than 50,000 cm1M1 at visible absorption maxima

Extremely low intrinsic uorescence, with quantum yields typically less than 0.01 when not bound to nucleic acids

Quantum yields typically greater than 0.4 when bound to nucleic acids

Available as blue-, green-, orange- or red-uorescent dyes, these novel SYTO stains provide researchers with visible lightexcitable dyes for labeling DNA and RNA in live cells (Figure 8.1.15). SYTO dyes dier from each other in one or more characteristics, including cell perme-ability, uorescence enhancement upon binding nucleic acids, excitation and emission spectra (Table 8.3), DNA/RNA selectivity and binding anity. e SYTO dyes are compatible with a variety of uorescence-based instruments that use either laser excitation or a conventional broadband illumination source (e.g., mercury- and xenon-arc lamps).

e SYTO dyes can stain both DNA and RNA. In most cases, the uorescence wave-lengths and emission intensities are similar for solution measurements of DNA or RNA bind-ing. Exceptions include the SYTO 12 and SYTO 14 dyes, which are about twice as uorescent when complexed with RNA as with DNA, and SYTO 16, which is about twice as uorescent on DNA than RNA. Consequently, the SYTO dyes do not act exclusively as nuclear stains in live cells and should not be equated in this regard with DNA-selective compounds such as DAPI or the Hoechst 33258 and Hoechst 33342 dyes, which readily stain cell nuclei at low concentrations in most cells. SYTO dyestained eukaryotic cells will generally show diuse cytoplasmic stain-ing, as well as nuclear staining. e SYTO 14 dye (S7576) has been used to visualize the trans-location of endogenous RNA found in polyribosome complexes in living cells.44,45 Particularly intense staining of intranuclear bodies is frequently observed. Because these dyes are generally cell permeant and most of the SYTO dyes contain a net positive charge at neutral pH, they may also stain mitochondria. In addition, the SYTO dyes will stain most gram-positive and








gram-negative bacterial cells. Dead yeast cells are brightly stained with the SYTO dyes, and live yeast cells typically exhibit staining of both the mitochondria and the nucleus. Some of the SYTO dyes have been reported to be useful for detecting apoptosis 46,47 (Section 15.5), and dyes structurally similar to the SYTO dyes have been used to detect multidrug-resistant cells 48 (Section 15.6). e red-uorescent SYTO dyes are proving useful as counterstains (Section 12.5) when combined with green-uorescent antibodies, lectins or the cell-impermeant SYTOX Green nucleic acid stain.

All of the SYTO dyes are available separately (Table 8.3), and several SYTO dyes are in-cluded in our LIVE/DEAD Viability Kits (Section 15.3, Table 15.2). e green-uorescent SYBR 14 dye, a component of our LIVE/DEAD Sperm Viability Kit (L7011, Section 15.3) is also in the SYTO family of dyes. To facilitate testing the SYTO dyes in new applications, we oer several sampler kits containing sample sizes of SYTO dyes in each color set (Table 8.3). e recom-mended dye concentration for cell staining depends on the assay and may vary widely but is typically 120 M for bacteria, 1100 M for yeast and 10 nM5 M for other eukaryotes.

SYTO RNASelect Green-Fluorescent Cell StainSYTO RNASelect green-uorescent cell stain (S32703, Section 15.2) is a cell-permeant

nucleic acid stain that selectively stains RNA (Figure 8.1.16, Figure 8.1.17). Although virtually nonuorescent in the absence of nucleic acids, the SYTO RNASelect stain exhibits bright green uorescence when bound to RNA (absorption/emission maxima ~490/530 nm), but only a weak uorescent signal when bound to DNA (Figure 8.1.16). Filter sets that are suitable for imag-ing cells labeled with uorescein (FITC) will work well for imaging cells stained with SYTO RNASelect stain (Figure 8.1.18).

Amine-Reactive Cyanine Dyee amine-reactive succinimidyl ester of SYBR 101 dye (S21500) can be conjugated to pep-

tides, proteins, drugs, polymeric matrices and biomolecules with primary amine groups. e conjugates are expected to be essentially nonuorescent until they complex with nucleic acids, resulting in strong green uorescence. us, they may be useful for studies of nucleic acid bind-ing to various biomolecules, such as DNA-binding proteins. It is also possible that the uores-cence enhancement upon nucleic acid binding of SYBR 101 dye conjugates will be useful for monitoring their transport into the nucleus. SYBR 101 dye conjugates of solid or semisolid matrices (such as microspheres, magnetic particles or various resins) may be useful for detection or anity isolation of nucleic acids.

e reactive SYBR 101 dye may also be conjugated to amine-modied nucleic acids. Although it is possible that the SYBR 101 dye may show some uorescence when conjugated to amine groups on nucleic acids, they may be useful for developing homogeneous hybridization assays in which a specic sequence can be quantitated in solution without the need to separate bound and free probes. For example, a similar reactive nucleic acid stain has been used to label pep-tidenucleic acid conjugates (PNA) for use as probes in real-time PCR. e labeled PNA probes exhibited a uorescence increase upon hybridization to their complementary sequence and have been used to identify a single-base mismatch in a 10-base target sequence.49,50

Figure 8.1.19 Ethidium bromide (15585-011). Figure 8.1.20 Propidium iodide (P1304MP).

Wavelength (nm)300 400 500

Ab

sorp

tion

600

RNADNAbuffer alone

Figure 8.1.16 Relative absorption (A) and uorescence emission (B) spectra of SYTO RNASelect green-uores-cent cell stain (S32703) in the presence of Escherichia coli DNA or in buer alone.

Wavelength (nm)450 550 650

Fluo

resc

ence

em

issi

on

700500 600

RNADNAbuffer alone

Figure 8.1.17 Methanol-xed bovine pulmonary artery endothelial cells treated with RNase, DNase or both, and then labeled with SYTO RNASelect Green cell stain (S32703). Removal of RNA with RNase prevented nucleolar labeling and greatly decreased nuclear and cytoplasmic la-beling. Use of DNase resulted in less of a loss of label inten-sity in these cell compartments, reecting the RNA-selective nature of this dye.

Treatment

Ave

rage

uo

resc

ence

inte

nsity

RNase + DNaseDNaseRNaseControl0

500

1000

1500

2000

2500

3000

Nucleolus

Nucleus

Cytoplasm

Figure 8.1.18 Methanol-xed MRC-5 cells stained with SYTO RNASelect green-uorescent cell stain (S32703). Nuclei were stained with DAPI (D1306, D3571, D21490); the densely stained areas are nucleoli.

A

B







Figure 8.1.22 Normalized uorescence emission spectra of DNA-bound 1) Hoechst 33258 (H1398, H3569, H21491), 2) acridine orange (A1301, A3568), 3) ethidium bromide (15585-011) and 4) 7-aminoactinomycin D (A1310).

Fluo

resc

ence

em

issi

on

1 2 3 4

Wavelength (nm)400 500 600 700

Figure 8.1.21 Day 10 of development of a Drosophila ovar-ian egg chamber assembly line. The nuclei of follicle and nurse cells were labeled with propidium iodide (P1304MP, P3566, P21493) and visualized by confocal laser-scanning microscopy using excitation by the 568 nm spectral line of an Ar-Kr laser. Image contributed by Sandra Orsulic, University of North Carolina at Chapel Hill.

Phenanthridines and Acridines: Classic Intercalating DyesCell-Impermeant Ethidium Bromide and Propidium Iodide

Ethidium bromide (EtBr, 15585-011; Figure 8.1.19) and propidium iodide (PI, P1304MP; P3566, P21493; Figure 8.1.20) are structurally similar phenanthridinium intercalators. PI is more soluble in water and less membrane-permeant than EtBr, although both dyes are generally ex-cluded from viable cells. EtBr and PI can be excited with mercury- or xenon-arc lamps or with the argon-ion laser, making them suitable for uorescence microscopy, confocal laser-scanning microscopy (Figure 8.1.21), ow cytometry and uorometry. ese dyes bind with little or no sequence preference at a stoichiometry of one dye per 45 base pairs of DNA.51 Excitation of the EtBrDNA complex may result in photobleaching of the dye and single-strand breaks.52 Both EtBr and PI also bind to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA. Once these dyes are bound to nucleic acids, their uorescence is enhanced ~10-fold, their excitation maxima are shied ~3040 nm to the red and their emission maxima are shied ~15 nm to the blue 53 (Figure 8.1.22, Table 8.4). Although their molar absorptivities (ex-tinction coecients) are relatively low, EtBr and PI exhibit suciently large Stokes shis to allow simultaneous detection of nuclear DNA and uorescein-labeled antibodies, provided that the proper optical lters are used.

PI is commonly used as a nuclear or chromosome counterstain (Section 12.5, Figure 8.1.21) and as a stain for dead cells (Section 15.2, Figure 8.1.23). EtBr is the conventional dye used for nucleic acid gel staining (Section 8.4). However, our SYBR Gold and SYBR Green nucleic acid gel stains are far more sensitive than EtBr, and the SYBR Green I stain has been shown to be signicantly less mutagenic than EtBr by Ames testing 54 (Section 8.4). Furthermore, our SYBR Safe DNA gel stain, which is as sensitive as EtBr and less mutagenic in the standard Ames test, has tested negative in three mammalian cellbased assays for genotoxicity and is not classied as hazardous waste under U.S. Federal regulations (Section 8.4).

EtBr and PI are potent mutagens and must be handled with extreme care. Solutions contain-ing EtBr or PI can be decontaminated by ltration through activated charcoal, which is then incinerated, thus providing an economical decontamination procedure.55 Alternatively, the dyes can be completely degraded in buer by reaction with sodium nitrite and hypophosphorous acid.56 PI is oered as a solid (P1304MP, P21493), and both EtBr and PI are available as aqueous solutions (15585-011, P3566).

Cell-Permeant Hexidium IodideHexidium iodide (H7593) is a moderately lipophilic phenanthridinium dye (Figure 8.1.24)

that is permeant to mammalian cells and selectively stains almost all gram-positive bacte-ria in the presence of gram-negative bacteria. Our LIVE BacLight Bacterial Gram Stain Kit and ViaGram Red+ Bacterial Gram Stain and Viability Kit (L7005, V7023; Section 15.3) use

Figure 8.1.23 A mixed population of live and isopro-pyl alcoholkilled Micrococcus luteus and Bacillus cereus stained with the LIVE/DEAD BacLight Bacterial Viability Kit (L7007, L7012). Bacteria with intact cell membranes ex-hibit green uorescence, whereas bacteria with damaged membranes exhibit red uorescence. Prior to imaging, the bacteria were placed onto a polycarbonate lter and im-mersed in BacLight mounting oil. This multiple exposure image was acquired with a triple-bandpass optical lter set appropriate for simultaneous imaging of DAPI, uorescein and Texas Red dyes.

Figure 8.1.24 Hexidium iodide (H7593).

Figure 8.1.25 A mixed population of Bacillus cereus and Pseudomonas aeruginosa stained with the dye mixture pro-vided in our LIVE BacLight Gram Stain Kit (L7005). When live bacteria are incubated with this kits cell-permeant nu-cleic acid stains, gram-positive organisms uoresce orange and gram-negative organisms uoresce green. The bacte-ria were photographed through an Omega Optical triple bandpass lter set.Figure 8.1.26 Dihydroethidium (hydroethidine, D1168).

N

H2N NH2

HCH2CH3








hexidium iodide for the discrimination of bacterial gram sign (Figure 8.1.25). Hexidium iodide yields slightly shorter-wavelength spectra upon DNA binding than our ethidium or propidium dyes. Generally, both the cytoplasm and nuclei of eukaryotic cells show staining with hexidium iodide; however, mitochondria and nucleoli may also be stained.

Cell-Permeant Dihydroethidium (Hydroethidine)Dihydroethidium (also known as hydroethidine) is a chemically reduced ethidium derivative

(Figure 8.1.26) that is permeant to live cells and exhibits blue uorescence in the cytoplasm. Many viable cells oxidize the probe to ethidium, which then uoresces red upon DNA intercalation 5759 (Figure 8.1.27). Dihydroethidium, which is somewhat air sensitive, is available in a 25 mg vial (D1168) or specially packaged in 10 vials of 1 mg each (D11347); the special packaging is strongly recommended when small quantities of the dye will be used at a time. Dihydroethidium is also available as a 5 mM stabilized solution in dimethylsulfoxide (D23107).

Figure 8.1.27 Live bovine pulmonary artery endothelial cells (BPAEC) were incubated with the cell-permeant, weakly blue-uorescent dihydroethidium (D1168, D11347, D23107) and the green-uorescent mitochondrial stain, MitoTracker Green FM (M7514). Upon oxidation, red-uorescent ethid-ium accumulated in the nucleus.

Figure 8.1.30 Absorption and uorescence emission spec-tra of ethidium homodimer-1 bound to DNA.

Figure 8.1.28 Ethidium homodimer-1 (EthD-1, E1169).

Figure 8.1.29 Ethidium homodimer-2 (EthD-2, E3599).

Table 8.4 Properties of classic nucleic acid stains.

Cat. No. Dye Ex/Em*Fluorescence Emission Color Applications

A666 Acridine homodimer

431/498 Green Impermeant AT-selective High-anity DNA binding

A1301A3568

Acridine orange 500/526 (DNA)460/650 (RNA)

Green/Red Permeant RNA/DNA discrimination measurements Lysosome labeling Flow cytometry Cell-cycle studies

A1310 7-AAD (7-amino-actinomycin D)

546/647 Red Weakly permeant GC-selective Flow cytometry Chromosome banding

A7592 Actinomycin D 442 None Chromosome banding

A1324 ACMA 419/483 Blue AT-selective Alternative to quinacrine for chromosome

Q banding Membrane phenomena

D1306D3571D21490

DAPI 358/461 Blue Semi-permeant AT-selective Cell-cycle studies Mycoplasma detection Chromosome and nuclei counterstain Chromosome banding

D1168D11347D23107

Dihydroethidium 518/605 Red Permeant Blue uorescent until oxidized to

ethidium

15585-011 Ethidium bromide 518/605 Red Impermeant dsDNA intercalator Dead-cell stain Chromosome counterstain Electrophoresis Flow cytometry Argon-ion laser excitable

E1169 Ethidium homodimer-1 (EthD-1)

528/617 Red Impermeant High-anity DNA labeling Dead-cell stain Electrophoresis prestain Argon-ion and green He-Ne laser

excitable

E3599 Ethidium homodimer-2 (EthD-2)

535/624 Red Impermeant Very high-anity DNA labeling Electrophoresis prestain

* Excitation (Ex) and emission (Em) maxima in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution, unless otherwise indicated. Indication of dyes as "permeant" or "impermeant" are for the most common applications; permeability to cell membranes may vary considerably with the cell type, dye concentrations and other staining conditions. Available in aqueous solution for those wishing to avoid potentially hazardous and mutagenic powders. After oxidation to ethidium. ** Prior to photolysis; after photolysis the spectra of the dye/DNA complexes are similar to those of ethidium bromideDNA complexes.







Cat. No. Dye Ex/Em*Fluorescence Emission Color Applications

E1374 Ethidium monoazide

464/625(unbound)**

Red Impermeant Photocrosslinkable Compatible with xation procedures

H7593 Hexidium iodide 518/600 Red Permeant, except gram-negative bacteria Stains nuclei and cytoplasm of eukaryotes

and some bacteria

H1398H3569H21491

Hoechst 33258 (bis-benzimide)

352/461 Blue Permeant AT-selective Minor groovebinding dsDNA-selective binding Cell-cycle studies Chromosome and nuclear counterstain

H1399H3570H21492

Hoechst 33342 350/461 Blue Permeant AT-selective Minor groovebinding dsDNA-selective binding Cell-cycle studies Chromosome and nuclear counterstain

H21486 Hoechst 34580 392/498 Blue Permeant AT-selective Minor groovebinding dsDNA-selective binding Cell-cycle studies Chromosome and nuclear counterstain

H22845 Hydroxy-stilbamidine

385/emission varies with nucleic acid

Varies AT-selective Spectra dependent on secondary

structure and sequence RNA/DNA discrimination Nuclear stain in tissue

L7595 LDS 751 543/712 (DNA)590/607 (RNA)

Red/infrared Permeant High Stokes shift Long-wavelength spectra Flow cytometry

N21485 Nuclear yellow 355/495 Yellow Impermeant Nuclear counterstain

P1304MPP3566P21493

Propidium iodide (PI)

530/625 Red Impermeant Dead-cell stain Chromosome and nuclear counterstain

* Excitation (Ex) and emission (Em) maxima in nm. All excitation and emission maxima were determined for dyes bound to double-stranded calf thymus DNA in aqueous solution, unless otherwise indicated. Indication of dyes as "permeant" or "impermeant" are for the most common applications; permeability to cell membranes may vary considerably with the cell type, dye concentrations and other staining conditions. Available in aqueous solution for those wishing to avoid potentially hazardous and mutagenic powders. After oxidation to ethidium. ** Prior to photolysis; after photolysis the spectra of the dye/DNA complexes are similar to those of ethidium bromideDNA complexes.

Table 8.4 Properties of classic nucleic acid stainscontinued.

High-Anity Ethidium HomodimersEthidium homodimer-1 (EthD-1, E1169; Figure 8.1.28) and ethidium homodimer-2 (EthD-

2, E3599; Figure 8.1.29) strongly bind to dsDNA, ssDNA, RNA and oligonucleotides with a signicant uorescence enhancement (>40-fold). EthD-1 also binds with high anity to triplex nucleic acid structures.60 One molecule of EthD-1 binds per four base pairs in dsDNA,4 and the dyes intercalation is not sequence selective.61 It was originally reported that only one of the two phenanthridinium rings of EthD-1 is bound at a time; 4 subsequent reports indicate that bis-intercalation appears to be involved in staining both double-stranded and triplex nucleic acids.20,60

e spectra and other properties of the EthD-1 and EthD-2 dimers are almost identical (Figure 8.1.30). However, the DNA anity of EthD-2 is about twice that of EthD-1. EthD-2 is also about twice as uorescent bound to dsDNA than to RNA. Because both EthD-1 and EthD-2 can be excited with UV light or by the 488 nm spectral line of the argon-ion laser, either dye can be used in combination with the TOTO-1, YOYO-1 or SYTOX Green nucleic acid stains for multicolor experiments (Figure 8.1.31). e ethidium homodimer dyes are impermeant to cells with intact membranes, a property that makes EthD-1 useful as a dead-cell indicator in our

Figure 8.1.31 Normalized uorescence emission spectra of DNA-bound SYTOX Green nucleic acid stain (S7020) and ethidium homodimer-1 (EthD-1, E1169). Both spectra were obtained using excitation at 488 nm.

Flu

ores

cenc

e em

issi

on

600

SYTOX Green EthD-1 Ex = 488 nm

500 700

Wavelength (nm)

Figure 8.1.32 Live and dead kangaroo rat (PtK2) cells stained with ethidium homodimer-1 and the esterase sub-strate calcein AM, both of which are provided in our LIVE/DEAD Viability/Cytotoxicity Kit (L3224). Live cells uoresce a bright green, whereas dead cells with compromised mem-branes uoresce red-orange.

Figure 8.1.33 A mixed population of trinitrophenyl-sen-sitized and nonsensitized bovine pulmonary artery endo-thelial cells that have been probed with antidinitrophenyl-KLH antibody (A6430), incubated with rabbit complement and then stained using the LIVE/DEAD Reduced Biohazard Viability/Cytotoxicity Kit #1 (L7013). With this kit, the mem-brane-permeant SYTO 10 dye labels the nucleic acids of live cells with green uorescence, and the membrane-imperme-ant DEAD Red dye labels nucleic acids of membrane-com-promised cells with red uorescence. Subsequent aldehyde-based xation inactivates pathogens without distorting the original staining pattern. This multiple-exposure image was acquired with bandpass optical lters appropriate for uo-rescein and Texas Red dyes.








LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Section 15.3, Figure 8.1.32) and EthD-2 (as Dead Red nucleic acid stain) a suitable dead-cell indicator in our LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 (L7013, Section 15.3, Figure 8.1.33). ese dyes have also been used to de-tect DNA in solution,61 although they are not as sensitive or as easy to use as our Quant-iT PicoGreen dsDNA reagent (Section 8.3).

Ethidium Monoazide: A Photocrosslinking ReagentNucleic acids can be covalently photolabeled by various DNA intercalators. Ethidium mono-

azide (E1374, Figure 8.1.34) is a uorescent photoanity label that, aer photolysis, binds co-valently to nucleic acids both in solution and in cells that have compromised membranes.6266 e quantum yield for covalent photolabeling by ethidium monoazide is unusually high (>0.4).

e membrane-impermeant ethidium monoazide is reported to only label dead cells and is therefore particularly useful for assaying the viability of pathogenic cells (Section 15.2). A mixed population of live and dead cells incubated with this reagent can be illuminated with a visible-light source, washed, xed and then analyzed in order to determine the viability of the cells at the time of photolysis.67 is method not only reduces some of the hazards inherent in working with pathogenic cells, but also is compatible with immunocytochemical analyses requiring xation. We have developed alternative assays for determining the original viability of xed samples and provide these in the LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 (L7013) and the LIVE/DEAD Fixable Dead Cell Stain Kits, which are described in Section 15.3.

In addition to its utility as a viability indicator, ethidium monoazide has been used to irre-versibly label the DNA of Candida albicans in order to investigate phagocytic capacity of leuko-cytes.68 Ethidium monoazide has also been employed to "footprint" drug-binding sites on DNA,69 to probe for ethidium-binding sites in DNA 70 and transfer RNA (tRNA) 65 and to selectively photoinactivate the expression of genes in vertebrate cells.71

Acridine Orange: A Dual-Fluorescence Nucleic Acid StainWe oer highly puried, ow cytometrygrade acridine orange, a dye that interacts with

DNA and RNA by intercalation or electrostatic attractions. In condensed chromatin, however, the bulk of DNA is packed in a way that does not allow ecient acridine orange intercalation.72 is cationic dye (Figure 8.1.35) has green uorescence with an emission maximum at 525 nm when bound to DNA. Upon association with RNA, its emission is shied to ~650 nm (red uo-rescence). Acr

chapter 8—nucleic acid detection and analysis

Documents