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PCR Detection of Viral DNA/RNA

In viral diagnostics, nucleic acid amplification tests, such as reverse transcription polymerase chain reaction (RT-PCR), permits the early detection and identification of infectious diseases known as retroviruses. In this modified version of the standard PCR process, mRNA, which serves as the initial template, is first reversed transcribed to complementary DNA (cDNA) and then subsequently amplified via PCR for downstream analysis. Relative to other techniques for measuring mRNA, such as Northern blot analysis, RNAse protection assays, or in situ hybridization, RT-PCR is significantly more robust at detecting the RNA transcript of any gene regardless of its relative abundance. RT-PCR has become an instrumental diagnostic tool for the detection of pathogens, including influenza viruses, enteroviruses, coronavirus (SARS-CoV-2), Ebola virus, and HIV.

 

 

Retroviruses


A retrovirus is a type of virus whose genetic material is encoded in RNA. Similar to other viruses, retroviruses hijack the cell's machinery in order to replicate copies of themselves. Upon infecting a cell, the retrovirus utilizes its own reverse-transcriptase enzyme to generate double-stranded DNA from its RNA genome. The newly synthesized viral DNA is integrated into the host cell genome, and the virus thereafter replicates as part of the host cell's DNA.

In humans, many retroviruses cause serious diseases. Retroviruses such as HIV-1 and HIV-2 cause the disease AIDS, while the human T-lymphotropic virus causes leukemia (i.e. cancer of the blood) and demyelinating disease. The severity of such diseases and the constant threat of new emerging infectious diseases has reaffirmed the need for developing rapid diagnostic tools capable of detecting low-density infections. One of the most well-established tools for the early detection of retroviral infection is nucleic acid amplification testing.

 

Nucleic Acid Amplification Tests


A nucleic acid amplification test is designed to detect small amounts of a particular nucleic acid sequence in order to identify a specific pathogen, often a virus or bacteria. Rather than detecting antigens or antibodies (the objective in serological analysis), nucleic acid amplification tests target and detect genetic materials, such as DNA or RNA. The detection of genetic materials allows for early diagnosis of infection prior to seroconversion, the time period during which a specific antibody develops against the pathogen and becomes detectable in the blood. Common nucleic acid amplification tests include reverse transcription polymerase chain reaction (RT-PCR), strand displacement assay (SDA), or transcription mediated assay (TMA).

Reverse Transcription PCR (RT-PCR)


Reverse transcription polymerase chain reaction (RT-PCR) is a highly sensitive technique for the detection and quantitation of mRNA expression levels. In RT-PCR, the RNA template is first reversed transcribed into complementary DNA (cDNA), using the enzyme reverse transcriptase. This RNA-dependent DNA polymerase, with the assistance of reverse transcription primers, deoxynucleotides (dNTPs: dATP, dTTP, dGTP and dCTP), catalyzes the synthesis of cDNA from its respective target RNA sequence. The cDNA is then used as a template for exponential amplification using standard PCR procedure (denaturation, annealing and elongation). RT-PCR is used in a variety of applications including gene expression analysis, microarray validation, pathogen detection, and disease research.

Quantification of RT-PCR products can be generally divided into two categories: end-point RT-PCR and quantitative RT-PCR (RT-qPCR).
 

Table 1. Deoxynucleotides (dNTPs) for use in PCR, real-time PCR, and reverse transcription PCR

Product
Solvent
Unit Size
Cat No.
ReadiUse™ dNTP Mix *10 mM*Water5 mL17200

 

End-point RT-PCR


End-point RT-PCR analysis, which is based on the plateau phase of PCR reaction, is used to analyze amplified products after all the cycles of the PCR reaction have been completed. In this method, amplicons are separated by agarose gel electrophoresis and visualized using a DNA binding dye, such as ethidium bromide (EtBr), to determine the size of the DNA molecules in the range of 500 to 30,000 bp. While EtBr is the most commonly used dye for visualizing DNA, it is mutagenic and highly toxic through inhalation. Instead, consider using safer and more environmentally friendly, non-toxic alternatives such as Gelite™ X100 (Cat No. 17706), Helixyte™ Green (Cat No. 17590), Helixyte™ Gold (Cat No. 17595), Gelite™ Green (Cat No. 17589) or Gelite™ Orange (Cat No. 17594).
 

Table 2. End-point RT-PCR detection methods For Quantifying RT-PCR products

End-Point RT-PCR
Overview
Relative RT-PCR
  • Utilizes primers for an internal control multiplexed with the gene specific primers
  • Requires compatible internal control and gene-specific primers
  • Can compare relative transcript abundance across multiple samples
  • Results are expressed as the ratio of gene signal to internal control signal, which can be used for the comparison between the samples in the estimation of relative target RNA expression
Competitive RT-PCR
  • Technique used for absolute quantification of a specific mRNA sequence in a sample.
  • Synthetic RNA and competitor RNA are added to sample RNA replicates and are co-amplified with the endogenous target
  • A concentration curve of the competitor RNA is produced and it is used to compare the RT-PCR signals produced from the endogenous transcripts to determine the amount of target present in the sample
Comparative RT-PCR
  • Similar to competitive RT-PCR in that the target RNA competes for amplification reagents within a single reaction with an internal standard of unrelated sequence
  • Results are compared to an external standard curve to determine the target RNA concentration
  • More convenient of the three methods as it does not require predetermination of mRNA (relative RT-PCR) or synthesis of a synthetic competitor RNA (competitive RT-PCR)

Table 3. Advantages, disadvantages and applications of end-point RT-PCR

End-Point RT-PCR
Advantages
  • Convenient - suitable for analysis of any double-strand DNA sequence
  • Economical - cheaper than using custom-designed probes
Disadvantages
  • Poor precision and low sensitivity
  • Short dynamic range <2 logs
  • Low resolution, size-based discrimination only
  • Not suitable for automation
  • Results are not expressed as numbers
  • Post-PCR processing
Applications
  • Gene expression analysis
  • Cloning
  • Constructing cDNA libraries
  • RNA sequencing
  • Microarray probe development
  • Species identification

Table 4. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis

Product
Ex (nm)¹
Filter²
Unit Size
Cat No.
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter1 mL17590
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter100 µL17604
Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution*254 mnLong path green filter1 mL17595
Gelite™ Green Nucleic Acid Gel Staining Kit254 nm or 300 nmLong path green filter1 Kit17589
Gelite™ Orange Nucleic Acid Gel Staining Kit254 nm or 300 nmLong path green filter1 Kit17594
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters100 µL17700
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters500 µL17701
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters1 mL17702
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters10 mL17703
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters100 µL17704
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters500 µL17705
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters1 mL17706
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution*254 nm, 300 nm or 520 nmEthidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters10 mL17707

 

Quantitative RT-PCR


In quantitative RT-PCR (RT-qPCR), fluorescent DNA-intercalating dyes or sequence-specific fluorescent probes are integrated into the RT-PCR reaction allowing for amplicon concentration to be measured in real-time during the exponential phase of PCR. By combining amplification and detection into a single-step, RT-qPCR provides greater precision and accuracy and produces quantitative data with a dynamic range several orders of magnitude larger than end-point RT-PCR. Because of its higher sensitivity, RT-qPCR is routinely used to analyze mRNA in gene expression, to examine the presence of retroviruses and to validate results obtained by array analyses.

Helixyte™ Green for RT-qPCR


RT-qPCR using fluorescent DNA-intercalating dyes, such as Helixyte™ Green (Cat No.17591) provides the easiest and most economical method for detecting and quantitating PCR amplicons in real-time. Helixyte™ Green binds to double-stranded DNA (dsDNA), and when excited emits light. As amplicon concentration increases with each successive cycle of amplification, so does the fluorescence intensity of Helixyte™ Green, to a degree proportional to the amount of dsDNA present in each PCR cycle. Helixyte™ Green is a much safer alternative than the highly mutagenic EtBr and can be used to monitor the amplification of any dsDNA sequence with greater sensitivity and less PCR inhibition. Because DNA-intercalating dyes will bind to any dsDNA, such as primer-dimers and non-specific products, it is important to use well-designed primers to avoid amplifying non-target sequences. To ensure amplification specificity and to check for primer-dimer artifacts, a melt curve analysis should be performed post-amplification.
 

Table 5. Double-stranded DNA-binding dyes for qPCR

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
Helixyte™ Green *20X Aqueous PCR Solution*498 nm522 nm5x1 mL17591
Helixyte™ Green *10,000X Aqueous PCR Solution*498 nm522 nm1 mL17592
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm1 mL17597
Helixyte™ Green dsDNA Quantifying Reagent *200X DMSO Solution*490 nm525 nm10 mL17598
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm100 µL17608
Q4ever™ Green *1250X DMSO Solution*503 nm527 nm2 mL17609


TaqMan® Probes and Molecular Beacons for RT-qPCR


In probe-based RT-qPCR, fluorescently-labeled, target-specific probes are used to measure DNA amplification in real-time. This method benefits from extreme specificity and affords the end-user the opportunity for multiplexing multiple targets in a single reaction. Of the many probe-based RT-qPCR chemistries available, TaqMan® probes and Molecular Beacons, are the most widely used and both depend upon Förster Resonance Energy Transfer (FRET) to generate a fluorescence signal. TaqMan® probes rely on the 5'-nuclease activity of Taq DNA polymerase. Short oligonucleotide sequences, complementary to the target of interest, are labeled with a fluorescent reporter dye at the 5' end (see Table 5 below) and a non-fluorescent quencher dye at the 3' end (see Table 6 below). During PCR cycling, primers and probe anneal to the target. As Taq DNA polymerase binds to and extends the primer upstream of the probe, the hybridized probe is hydrolyzed and the fragment containing the reporter dye is released. The fluorescence signal can now be detected and the amount of fluorescence signal generated is proportional to the amount of qPCR products produced.

Like TaqMan® probes, Molecular Beacons are labeled with a fluorescent reporter dye at the 5' end and a non-fluorescent quencher dye at the 3' end. However, this method does not rely on the 5' nuclease activity of Taq DNA polymerase to generate a signal, rather Molecular Beacons are designed to remain intact during the entire amplification process. In the absence of the target, Molecular Beacons remain in a 'hairpin' confirmation due to its self-complementary stem structure. This brings both the fluorescent reporter and quencher dyes within close proximity of one another preventing the probe from fluorescing. When the Molecular Beacon hybridizes to its target, the fluorescent reporter and the quencher are separated, and the reporter dye emits at its characteristic wavelength.

We offer a broad range of dye phosphoramidites and dye CPG supports for developing FRET oligonucleotides, TaqMan® probes and Molecular Beacons. This set includes classic dyes such as FAM, HEX, TET and JOE, as well as superior oligo-labeling dyes such as Tide Fluor™ and Tide Quencher™ dyes.
 

Table 6. Fluorescent reporter dyes for labeling the 5' end or 3' end on sequence-specific qPCR probes.

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] *CAS 50402-56-7*3364551 g610
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] *CAS 50402-56-7*33645510 g611
EDANS C5 maleimide3364555 mg619
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0*3364551 g615
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] *CAS 100900-07-0*33645510 g616
Tide Fluor™ 1 acid [TF1 acid] *Superior replacement for EDANS*341448100 mg2238
Tide Fluor™ 1 alkyne [TF1 alkyne]3414485 mg2237
Tide Fluor™ 1 amine [TF1 amine] *Superior replacement for EDANS*3414485 mg2239
Tide Fluor™ 1 azide [TF1 azide]3414485 mg2236
Tide Fluor™ 1 CPG [TF1 CPG] *500 Å*341448100 mg2240
Tide Fluor™ 1 CPG [TF1 CPG] *1000 Å*341448100 mg2241
Tide Fluor™ 1 maleimide [TF1 maleimide] *Superior replacement for EDANS*3414485 mg2242
Tide Fluor™ 1 succinimidyl ester [TF1 SE] *Superior replacement for EDANS*3414485 mg2244
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*4935171 g100
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*49351710 g101
5(6)-FAM [5-(and-6)-Carboxyfluorescein] *CAS 72088-94-9*49351725 g102
5(6)-FAM cadaverine493517100 mg127
5(6)-FAM ethylenediamine493517100 mg123
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*49351725 mg110
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*493517100 mg111
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] *CAS 117548-22-8*4935171 g112
6-FAM [6-Carboxyfluorescein]493517100 mg106
6-FAM [6-Carboxyfluorescein]4935171 g107
6-FAM [6-Carboxyfluorescein]4935175 g108
6-FAM Alkyne49351710 mg134
6-FAM Alkyne493517100 mg956
6-FAM Azide49351710 mg133
6-FAM Azide493517100 mg955
FAM-xtra™ Phosphoramidite 49351750 µmoles6037
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]493517100 µmoles6016
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]49351710x100 µmoles6017
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*49351710 mg116
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*493517100 mg117
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] *CAS 92557-81-8*4935171 g118
6-Fluorescein phosphoramidite498517100 µmoles6018
6-Fluorescein phosphoramidite49851710x100 µmoles6019
3'-(6-Fluorescein) CPG *1000 Å*4985171 g6014
Tide Fluor™ 2 acid [TF2 acid] *Superior replacement for fluorescein*50352525 mg2245
Tide Fluor™ 2 alkyne [TF2 alkyne] *Superior replacement for fluorescein*5035251 mg2253
Tide Fluor™ 2 amine [TF2 amine] *Superior replacement for fluorescein*5035251 mg2246
Tide Fluor™ 2 azide [TF2 azide] *Superior replacement for fluorescein*5035251 mg2252
Tide Fluor™ 2 maleimide [TF2 maleimide] *Superior replacement for fluorescein*5035251 mg2247
Tide Fluor™ 2, succinimidyl ester [TF2 SE] *Superior replacement for fluorescein*5035255 mg2248
Tide Fluor™ 2WS acid [TF2WS acid] *Superior replacement for FITC*50352510 mg2348
Tide Fluor™ 2WS amine [TF2WS amine] *Superior replacement for FITC*5035251 mg2351
Tide Fluor™ 2WS maleimide [TF2WS maleimide] *Superior replacement for FITC*5035251 mg2350
Tide Fluor™ 2WS succinimidyl ester [TF2WS SE] *Superior replacement for FITC*5035255 mg2349
6-TET alkyne5215435 mg245
6-TET azide5215435 mg244
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]52154350 µmoles6021
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]521543100 µmoles6027
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]52154310x100 µmoles6025
6-TET, SE [6-Carboxy-2',4,7',7-tetrachlorofluorescein, succinimidyl ester]5215435 mg211
Helix Fluor™ 545, succinimidyl ester5265431 mg250
VIC phosphoramidite52654350 µmoles6080
VIC phosphoramidite526543100 µmoles6081
VIC phosphoramidite5265431 g6082
5-VIC phosphoramidite52654350 µmoles6083
5-VIC phosphoramidite526543100 µmoles6084
5-VIC phosphoramidite5265431 g6085
6-VIC, SE [6-VIC NHS ester]5265431 mg212
6-VIC, SE [6-VIC NHS ester]5265435 mg213
6-HEX alkyne5335595 mg241
6-HEX azide5335595 mg240
6-HEX, SE [6-Carboxy-2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester]5335595 mg202
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]533559100 µmoles6026
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]53355910x100 µmoles6024
6-NED alkyne5455671 mg216
6-NED azide5455671 mg217
6-NED maleimide5455671 mg218
6-NED, SE [6-NED NHS ester]5455671 mg214
6-NED, SE [6-NED NHS ester]5455671 mg215
Helix Fluor™ 575, succinimidyl ester5535701 mg251
Tide Fluor™ 3 acid [TF3 acid] *Superior replacement for Cy3*54657125 mg2268
Tide Fluor™ 3 alkyne [TF3 alkyne] *Superior replacement for Cy3*5465711 mg2255
Tide Fluor™ 3 amine [TF3 amine] *Superior replacement for Cy3*5465711 mg2269
Tide Fluor™ 3 azide [TF3 azide] *Superior replacement for Cy3*5465711 mg2254
Tide Fluor™ 3 maleimide [TF3 maleimide] *Superior replacement for Cy3*5465711 mg2270
Tide Fluor™ 3 succinimidyl ester [TF3 SE] *Superior replacement for Cy3*5465711 mg2271
Tide Fluor™ 3 phosphoramidite [TF3 CEP] *Superior replacement to Cy3 phosphoramidite*546571100 µmoles2274
Tide Fluor™ 3WS acid [TF3WS acid] *Superior replacement for Cy3*55156310 mg2268
Tide Fluor™ 3WS amine [TF3 amine] *Superior replacement for Cy3*5515631 mg2347
Tide Fluor™ 3WS maleimide [TF3 maleimide] *Superior replacement for Cy3*5515631 mg2344
Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] *Superior replacement for Cy3*5515631 mg2346
Tide Fluor™ 4 acid [TF4 acid] *Superior replacement for ROX and Texas Red*57860210 mg2285
Tide Fluor™ 4 alkyne [TF4 alkyne] *Superior replacement for ROX and Texas Red*5786021 mg2301
Tide Fluor™ 4 amine [TF4 amine] *Superior replacement for ROX and Texas Red*5786021 mg2286
Tide Fluor™ 4 azide [TF4 azide] *Superior replacement for ROX and Texas Red*5786021 mg2300
Tide Fluor™ 4 maleimide [TF4 maleimide] *Superior replacement for ROX and Texas Red*5786021 mg2287
Tide Fluor™ 4, succinimidyl ester [TF4 SE] *Superior replacement for ROX and Texas Red*5786025 mg2289
Tide Fluor™ 5WS acid [TF5WS acid] *Superior replacement for Cy5*64966410 mg2278
Tide Fluor™ 5WS alkyne [TF5WS alkyne] *Superior replacement for Cy5*6496641 mg2276
Tide Fluor™ 5WS amine [TF5WS amine] *Superior replacement for Cy5*6496641 mg2279
Tide Fluor™ 5WS azide [TF5WS azide] *Superior replacement for Cy5*6496641 mg2275
Tide Fluor™ 5WS maleimide [TF5WS maleimide] *Superior replacement for Cy5*6496641 mg2280
Tide Fluor™ 5WS succinimidyl ester [TF5WS SE] *Superior replacement for Cy5*6496645 mg2281
Tide Fluor™ 6WS acid [TF6WS acid] *Superior replacement for Cy5.5*68270110 mg2291
Tide Fluor™ 6WS alkyne [TF6WS alkyne] *Superior replacement for Cy5.5*6827011 mg2303
Tide Fluor™ 6WS amine [TF6WS amine] *Superior replacement for Cy5.5*6827011 mg2292
Tide Fluor™ 6WS azide [TF6WS azide] *Superior replacement for Cy5.5*6827011 mg2302
Tide Fluor™ 6WS maleimide [TF6WS maleimide] *Superior replacement for Cy5.5*6827011 mg2293
Tide Fluor™ 6WS succinimidyl ester [TF6WS SE] *Superior replacement for Cy5.5*6827011 mg2294
Tide Fluor™ 7WS acid [TF7WS acid] *Superior replacement for Cy7*75678010 mg2330
Tide Fluor™ 7WS alkyne [TF7WS alkyne] *Superior replacement for Cy7*7567801 mg2305
Tide Fluor™ 7WS amine [TF7WS amine] *Superior replacement for Cy7*7567801 mg2331
Tide Fluor™ 7WS azide [TF7WS azide] *Superior replacement for Cy7*7567801 mg2304
Tide Fluor™ 7WS maleimide [TF7WS maleimide] *Superior replacement for Cy7*7567801 mg2332
Tide Fluor™ 7WS succinimidyl ester [TF7WS SE] *Superior replacement for Cy7*7567801 mg2333
Tide Fluor™ 8WS acid [TF8WS acid] *Near Infrared Emission*78580110 mg2335
Tide Fluor™ 8WS alkyne [TF8WS alkyne] *Near Infrared Emission*7858011 mg2307
Tide Fluor™ 8WS amine [TF8WS amine] *Near Infrared Emission*7858011 mg2336
Tide Fluor™ 8WS azide [TF8WS azide] *Near Infrared Emission*7858011 mg2306
Tide Fluor™ 8WS maleimide [TF8WS maleimide] *Near Infrared Emission*7858011 mg2337
Tide Fluor™ 8WS succinimidyl ester [TF8WS SE] *Near Infrared Emission*7858011 mg2338

Table 7. Quencher dyes for labeling the 5' end or 3' end on sequence-specific qPCR probes.

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
DABCYL acid [4-((4-(Dimethylamino)phenyl)azo)benzoic acid] *CAS 6268-49-1*454N/A5 g2001
DABCYL C2 amine454N/A100 mg2006
DABCYL C2 maleimide454N/A25 mg2008
DABCYL-DBCO454N/A5 mg2010
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4*454N/A1 g2004
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] *CAS 146998-31-4*454N/A5 g2005
3'-DABCYL CPG *1000 Å*454N/A1 g6008
5'-DABCYL C6 Phosphoramidite454N/A1 g6009
Tide Quencher™ 1 acid [TQ1 acid]492N/A100 mg2190
Tide Quencher™ 1 alkyne [TQ1 alkyne]492N/A5 mg2189
Tide Quencher™ 1 amine [TQ1 amine]492N/A5 mg2192
Tide Quencher™ 1 azide [TQ1 azide]492N/A5 mg2188
Tide Quencher™ 1 CPG [TQ5 CPG] *500 Å*492N/A100 mg2193
Tide Quencher™ 1 CPG [TQ5 CPG] *1000 Å*492N/A100 mg2194
Tide Quencher™ 1 maleimide [TQ1 maleimide]492N/A5 mg2196
Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]492N/A100 µmoles2198
Tide Quencher™ 1 succinimidyl ester [TQ1 SE]492N/A25 mg2199
Tide Quencher™ 2 acid [TQ2 acid]516N/A100 mg2200
Tide Quencher™ 2 alkyne [TQ2 alkyne]516N/A5 mg2212
Tide Quencher™ 2 amine [TQ2 amine]516N/A5 mg2202
Tide Quencher™ 2 azide [TQ2 azide]516N/A5 mg2211
Tide Quencher™ 2 CPG [TQ5 CPG] *500 Å*516N/A100 mg2203
Tide Quencher™ 2 CPG [TQ5 CPG] *1000 Å*516N/A100 mg2204
Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]516N/A100 µmoles2208
Tide Quencher™ 2 succinimidyl ester [TQ2 SE]516N/A25 mg2210
Tide Quencher™ 2WS acid [TQ2WS acid]516N/A25 mg2050
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]516N/A1 mg2213
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]516N/A5 mg2214
Tide Quencher™ 2WS maleimide [TQ2WS maleimide]516N/A1 mg2059
Tide Quencher™ 2WS succinimidyl ester [TQ2WS, SE]516N/A5 mg2058
BXQ-1 Alkyne522N/A1 mg2414
BXQ-1 Amine522N/A5 mg2406
BXQ-1 Azide522N/A1 mg2412
BXQ-1 Carboxylic Acid522N/A25 mg2400
BXQ-1 CPG (500 Å)522N/A100 mg2408
BXQ-1 CPG (1000 Å)522N/A100 mg2410
BXQ-1 Maleimide522N/A1 mg2404
BXQ-1 Succinimidyl Ester522N/A5 mg2402
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*55257810 mg363
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*552578100 mg364
5-TAMRA [5-Carboxytetramethylrhodamine] *CAS 91809-66-4*5525781 g365
Rhodamine aldehyde [5-TAMRA aldehyde]5525785 mg9005
5-TAMRA alkyne5525785 mg487
5-TAMRA alkyne55257850 mg961
5-TAMRA azide5525785 mg486
5-TAMRA azide55257850 mg960
5-TAMRA cadaverine5525785 mg356
5-TAMRA ethylenediamine5525785 mg358
5-TAMRA C6 maleimide5525785 mg424
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*5525785 mg373
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*552578100 mg374
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-68-7*5525781 g375
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*552578100 mg360
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*5525781 g361
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] *CAS 98181-63-6*5525785 g362
5(6)-TAMRA cadaverine55257825 mg355
5(6)-TAMRA ethylenediamine55257825 mg354
5(6)-TAMRA Maleimide [Tetramethylrhodamine-5-(and-6)-maleimide]5525785 mg412
5(6)-TAMRA C6 maleimide5525785 mg423
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*55257825 mg370
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*552578100 mg371
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] *CAS 246256-50-8*5525781 g372
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*55257810 mg366
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*552578100 mg367
6-TAMRA [6-Carboxytetramethylrhodamine] *CAS 91809-67-5*5525781 g368
6-TAMRA alkyne5525785 mg491
6-TAMRA alkyne55257850 mg966
6-TAMRA azide5525785 mg490
6-TAMRA azide55257850 mg965
6-TAMRA cadaverine5525785 mg357
6-TAMRA CPG *1000 Å*5525781 g6051
6-TAMRA ethylenediamine5525785 mg359
6-TAMRA Maleimide [Tetramethylrhodamine-6-maleimide] *CAS 174568-68-4*5525781 mg419
6-TAMRA C6 maleimide5525785 mg425
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*5525785 mg376
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*552578100 mg377
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] *CAS#: 150810-69-8*5525781 g378
BXQ-2 Alkyne554N/A1 mg2434
BXQ-2 Amine554N/A5 mg2426
BXQ-2 Azide554N/A1 mg2432
BXQ-2 Carboxylic Acid554N/A25 mg2420
BXQ-2 CPG (500 Å)554N/A100 mg2428
BXQ-2 CPG (1000 Å)554N/A100 mg2430
BXQ-2 Maleimide554N/A1 mg2424
BXQ-2 Succinimidyl Ester554N/A5 mg2422
Tide Quencher™ 3 acid [TQ3 acid]573N/A100 mg2220
Tide Quencher™ 3 alkyne [TQ3 alkyne]573N/A5 mg2232
Tide Quencher™ 3 amine [TQ3 amine]573N/A5 mg2222
Tide Quencher™ 3 azide [TQ3 azide]573N/A5 mg2231
Tide Quencher™ 3 CPG [TQ5 CPG] *500 Å*573N/A100 mg2223
Tide Quencher™ 3 CPG [TQ5 CPG] *1000 Å*573N/A100 mg2224
Tide Quencher™ 3 maleimide [TQ3 maleimide]573N/A5 mg2226
Tide Quencher™ 3 phosphoramidite [TQ3 phosphoramidite]573N/A100 µmoles2228
Tide Quencher™ 3 succinimidyl ester [TQ3 SE]573N/A25 mg2230
Tide Quencher™ 3WS acid [TQ3WS acid]573N/A1 mg2229
Tide Quencher™ 3WS succinimidyl ester [TQ3WS SE]573N/A5 mg2227
Tide Quencher™ 4 CPG [TQ5 CPG] *500 Å*603N/A100 mg2062
Tide Quencher™ 4 CPG [TQ5 CPG] *1000 Å*603N/A100 mg2063
Tide Quencher™ 4WS acid [TQ4WS acid]603N/A5 mg2060
Tide Quencher™ 4WS alkyne [TQ4WS alkyne]603N/A1 mg2069
Tide Quencher™ 4WS amine [TQ4WS amine]603N/A1 mg2061
Tide Quencher™ 4WS azide [TQ4WS azide]603N/A1 mg2068
Tide Quencher™ 4WS maleimide [TQ4WS maleimide]603N/A1 mg2064
Tide Quencher™ 4WS succinimidyl ester [TQ4WS SE]603N/A1 mg2067
Tide Quencher™ 5 CPG [TQ5 CPG] *500 Å*661N/A100 mg2077
Tide Quencher™ 5 CPG [TQ5 CPG] *1000 Å*661N/A100 mg2078
Tide Quencher™ 5WS acid [TQ5WS acid]661N/A5 mg2075
Tide Quencher™ 5WS alkyne [TQ5WS alkyne]661N/A1 mg2083
Tide Quencher™ 5WS amine [TQ5WS amine]661N/A1 mg2076
Tide Quencher™ 5WS azide [TQ5WS azide]661N/A1 mg2082
Tide Quencher™ 5WS maleimide [TQ5WS maleimide]661N/A1 mg2079
Tide Quencher™ 5WS succinimidyl ester [TQ5WS SE]661N/A1 mg2081
Tide Quencher™ 6WS acid [TQ6WS acid]694N/A5 mg2090
Tide Quencher™ 6WS alkyne [TQ6WS alkyne]694N/A1 mg2098
Tide Quencher™ 6WS amine [TQ6WS amine]694N/A1 mg2091
Tide Quencher™ 6WS azide [TQ6WS azide]694N/A1 mg2097
Tide Quencher™ 6WS maleimide [TQ6WS maleimide]694N/A1 mg2094
Tide Quencher™ 6WS succinimidyl ester [TQ6WS SE]694N/A1 mg2096
Tide Quencher™ 7WS acid [TQ7WS acid]764N/A5 mg2105
Tide Quencher™ 7WS alkyne [TQ7WS alkyne]764N/A1 mg2113
Tide Quencher™ 7WS amine [TQ7WS amine]764N/A1 mg2106
Tide Quencher™ 7WS azide [TQ7WS azide]764N/A1 mg2112
Tide Quencher™ 7WS maleimide [TQ7WS maleimide]764N/A1 mg2109
Tide Quencher™ 7WS succinimidyl ester [TQ7WS SE]764N/A1 mg2111

Table 8. Recommended FRET pairs for developing FRET oligonucleotides

Donor \ Acceptor
DABCYL
TQ1
TQ2
TQ3
TQ4
TQ5
TQ6
TQ7
EDANS+++++++-----
MCA+++++++-----
Tide Fluor™ 1+++++++-----
FAM
FITC
++++++----
Cy2®
Tide Fluor™ 2
++++++----
HEX
JOE
TET
--+++++---
Cy3®
TAMRA
Tide Fluor™ 3
--+++++---
ROX
Texas Red®
---+++++--
Tide Fluor™ 4---+++++--
Cy5®
Tide Fluor™ 5
----+++++-
Cy5.5®
Tide Fluor™ 6
-----+++++
Cy7®
Tide Fluor™ 7
------++++

 

Tide Fluor™ Dyes for Labeling Oligos and Peptides


Tide Fluor™ dyes are a series of donor dyes optimized for developing FRET oligonucleotides and peptides for a variety of biological applications. Compared to common donor dyes such as EDANS, FAM, TAMRA, ROX, Cy 3 and Cy5, Tide Fluor™ dyes exhibit stronger fluorescence and higher photostability. They are the best affordable fluorescent dyes for labeling peptides and oligonucleotides without sacrificing performance.

Learn More

 

Table 9. Tide Fluor™ Dyes and Spectral Properties For Quantifying RT-PCR products

Labeling Dye
Abs (nm)
Em (nm)
ε1
Φ2
CF at 260 nm3
CF at 280 nm4
Tide Fluor™ 1345442200000.950.2460.187
Tide Fluor™ 2WS502525750000.90.2110.091
Tide Fluor™ 2500527750000.90.2880.201
Tide Fluor™ 3WS5555651500000.1050.0790.079
Tide Fluor™ 3555584850000.850.3310.201
Tide Fluor™ 4590618900000.910.4890.436
Tide Fluor™ 5WS6496642500000.250.0230.027
Tide Fluor™ 6WS6766952200000.180.1110.009
Tide Fluor™ 7WS7497752750000.120.0090.049
Tide Fluor™ 8WS7758072500000.080.1030.109
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. Φ = Fluorescence quantum yield in aqueous buffer (pH 7.2).
  3. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligos and nucleic acid labeling).
  4. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptide and protein labeling).

 

Tide Quencher™ Dyes for Labeling Oligos and Peptides


TF and TQ Probes
Hybridization-induced fluorescence enhancement of Molecular Beacon oligonucleotide probes that contain Tide Fluor™ dyes as donor or Tide Quencher™ dyes as acceptors.
Although DABCYL has been used to develop a variety of FRET applications, its low quenching efficiency for longer wavelength dyes (such as fluoresceins, rhodamines and cyanines) has limited its use in the development of sensitive fluorogenic FRET probes. Additionally, the absorption spectrum of DABCYL is environment-sensitive. AAT Bioquest has developed robust Tide Quencher™ acceptor dyes for the development of longer wavelength FRET probes. Tide Quencher™ dyes are a great choice to eliminate the limitations of classic quenchers.

The Key Benefits of Tide Quencher™ Dyes:

  • Explore the FRET potentials that might be impossible with other quenchers.
  • Versatile reactive forms are convenient for self-constructing your desired FRET biomolecules.
  • Perfectly match your desired fluorescent donors.
  • Competitive price with better performance.

 

Table 10. Tide Quencher™ Dyes and Spectral Properties For Quantifying RT-PCR products

Quencher
Abs (nm)
ε*1
CF at 260 nm*2
CF at 280 nm*3
Tide Quencher™ 148820,0000.1470.194
Tide Quencher™ 251221,0000.1000.120
Tide Quencher™ 2WS51521,0000.1000.120
Tide Quencher™ 357622,0000.0850.091
Tide Quencher™ 3WS57690,0000.1860.205
Tide Quencher™ 460423,0000.1460.183
Tide Quencher™ 4WS60490,0000.1490.136
Tide Quencher™ 566124,0000.1700.082
Tide Quencher™ 5WS661130,0000.0720.082
Tide Quencher™ 6WS691130,0000.1200.102
Tide Quencher™ 7WS759140,0000.0720.091
  1. ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm-1M-1).
  2. CF at 260 nm is the correction factor used for eliminating the dye contribution to the absorbance at 260 nm (for oligos and nucleic acid labeling).
  3. CF at 280 nm is the correction factor used for eliminating the dye contribution to the absorbance at 280 nm (for peptide and protein labeling).