PCR Detection of Viral DNA/RNA

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Polymerase Chain Reaction (PCR)
Digital PCR
PCR Detection of Viral DNA/RNA
ROX Reference Dye for Real-Time PCR

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
Nucleic Acid Amplification Tests

2.1 Reverse Transcription PCR (RT-PCR)

End-point RT-PCR
Quantitative RT-PCR

4.1 Helixyte™ Green for RT-qPCR
4.2 TaqMan® Probes and Molecular Beacons for RT-qPCR

Tide Fluor™ Dyes for Labeling Oligos and Peptides
Tide Quencher™ Dyes for Labeling Oligos and Peptides

6.1 The Key Benefits of Tide Quencher™ Dyes:

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	Water	5 mL	17200
ReadiUse™ ddNTP Terminator Mix 10 mM	Water	100 nmoles	17205
ReadiUse™ dNTP Mix Set 10 mM PCR Grade	Water	1 mL	17258

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 mn	Long path green filter	1 mL	17590
Helixyte™ Green Nucleic Acid Gel Stain 10,000X DMSO Solution	254 mn	Long path green filter	100 µL	17604
Helixyte™ Gold Nucleic Acid Gel Stain 10,000X DMSO Solution	254 mn	Long path green filter	1 mL	17595
Gelite™ Green Nucleic Acid Gel Staining Kit	254 nm or 300 nm	Long path green filter	1 Kit	17589
Gelite™ Orange Nucleic Acid Gel Staining Kit	254 nm or 300 nm	Long path green filter	1 Kit	17594
Gelite™ Safe DNA Gel Stain 10,000X Water Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	100 µL	17700
Gelite™ Safe DNA Gel Stain 10,000X Water Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	500 µL	17701
Gelite™ Safe DNA Gel Stain 10,000X Water Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	1 mL	17702
Gelite™ Safe DNA Gel Stain 10,000X Water Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	10 mL	17703
Gelite™ Safe DNA Gel Stain 10,000X DMSO Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	100 µL	17704
Gelite™ Safe DNA Gel Stain 10,000X DMSO Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	500 µL	17705
Gelite™ Safe DNA Gel Stain 10,000X DMSO Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	1 mL	17706
Gelite™ Safe DNA Gel Stain 10,000X DMSO Solution	254 nm, 300 nm or 520 nm	Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters	10 mL	17707

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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 nm	522 nm	5x1 mL	17591
Helixyte™ Green 10,000X Aqueous PCR Solution	498 nm	522 nm	1 mL	17592
Helixyte™ Green dsDNA Quantifying Reagent 200X DMSO Solution	490 nm	525 nm	1 mL	17597
Helixyte™ Green dsDNA Quantifying Reagent 200X DMSO Solution	490 nm	525 nm	10 mL	17598
Q4ever™ Green 1250X DMSO Solution	503 nm	527 nm	100 µL	17608
Q4ever™ Green 1250X DMSO Solution	503 nm	527 nm	2 mL	17609

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	336	455	1 g	610
EDANS acid [5-((2-Aminoethyl)amino)naphthalene-1-sulfonic acid] CAS 50402-56-7	336	455	10 g	611
EDANS C5 maleimide	336	455	5 mg	619
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] CAS 100900-07-0	336	455	1 g	615
EDANS sodium salt [5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid, sodium salt] CAS 100900-07-0	336	455	10 g	616
Tide Fluor™ 1 acid [TF1 acid] Superior replacement for EDANS	341	448	100 mg	2238
Tide Fluor™ 1 alkyne [TF1 alkyne]	341	448	5 mg	2237
Tide Fluor™ 1 amine [TF1 amine] Superior replacement for EDANS	341	448	5 mg	2239
Tide Fluor™ 1 azide [TF1 azide]	341	448	5 mg	2236
Tide Fluor™ 1 CPG [TF1 CPG] 500 Å	341	448	100 mg	2240
Tide Fluor™ 1 CPG [TF1 CPG] 1000 Å	341	448	100 mg	2241
Tide Fluor™ 1 maleimide [TF1 maleimide] Superior replacement for EDANS	341	448	5 mg	2242
Tide Fluor™ 1 succinimidyl ester [TF1 SE] Superior replacement for EDANS	341	448	5 mg	2244
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	1 g	100
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	10 g	101
5(6)-FAM [5-(and-6)-Carboxyfluorescein] CAS 72088-94-9	493	517	25 g	102
5(6)-FAM cadaverine	493	517	100 mg	127
5(6)-FAM ethylenediamine	493	517	100 mg	123
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	25 mg	110
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	100 mg	111
5(6)-FAM, SE [5-(and-6)-Carboxyfluorescein, succinimidyl ester] CAS 117548-22-8	493	517	1 g	112
6-FAM [6-Carboxyfluorescein]	493	517	100 mg	106
6-FAM [6-Carboxyfluorescein]	493	517	1 g	107
6-FAM [6-Carboxyfluorescein]	493	517	5 g	108
6-FAM Alkyne	493	517	10 mg	134
6-FAM Alkyne	493	517	100 mg	956
6-FAM Azide	493	517	10 mg	133
6-FAM Azide	493	517	100 mg	955
FAM-xtra™ Phosphoramidite	493	517	50 µmoles	6037
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]	493	517	100 µmoles	6016
6-FAM phosphoramidite [5'-Fluorescein phosphoramidite]	493	517	10x100 µmoles	6017
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] CAS 92557-81-8	493	517	10 mg	116
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] CAS 92557-81-8	493	517	100 mg	117
6-FAM, SE [6-Carboxyfluorescein, succinimidyl ester] CAS 92557-81-8	493	517	1 g	118
6-Fluorescein phosphoramidite	498	517	100 µmoles	6018
6-Fluorescein phosphoramidite	498	517	10x100 µmoles	6019
3'-(6-Fluorescein) CPG 1000 Å	498	517	1 g	6014
Tide Fluor™ 2 acid [TF2 acid] Superior replacement for fluorescein	503	525	25 mg	2245
Tide Fluor™ 2 alkyne [TF2 alkyne] Superior replacement for fluorescein	503	525	1 mg	2253
Tide Fluor™ 2 amine [TF2 amine] Superior replacement for fluorescein	503	525	1 mg	2246
Tide Fluor™ 2 azide [TF2 azide] Superior replacement for fluorescein	503	525	1 mg	2252
Tide Fluor™ 2 maleimide [TF2 maleimide] Superior replacement for fluorescein	503	525	1 mg	2247
Tide Fluor™ 2, succinimidyl ester [TF2 SE] Superior replacement for fluorescein	503	525	5 mg	2248
Tide Fluor™ 2WS acid [TF2WS acid] Superior replacement for FITC	503	525	10 mg	2348
Tide Fluor™ 2WS amine [TF2WS amine] Superior replacement for FITC	503	525	1 mg	2351
Tide Fluor™ 2WS maleimide [TF2WS maleimide] Superior replacement for FITC	503	525	1 mg	2350
Tide Fluor™ 2WS succinimidyl ester [TF2WS SE] Superior replacement for FITC	503	525	5 mg	2349
6-TET alkyne	521	543	5 mg	245
6-TET azide	521	543	5 mg	244
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]	521	543	50 µmoles	6021
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]	521	543	100 µmoles	6027
6-TET phosphoramidite [5'-Tetrachlorofluorescein phosphoramidite]	521	543	10x100 µmoles	6025
6-TET, SE [6-Carboxy-2',4,7',7-tetrachlorofluorescein, succinimidyl ester]	521	543	5 mg	211
Helix Fluor™ 545, succinimidyl ester	526	543	1 mg	250
VIC phosphoramidite	526	543	50 µmoles	6080
VIC phosphoramidite	526	543	100 µmoles	6081
VIC phosphoramidite	526	543	1 g	6082
5-VIC phosphoramidite	526	543	50 µmoles	6083
5-VIC phosphoramidite	526	543	100 µmoles	6084
5-VIC phosphoramidite	526	543	1 g	6085
6-VIC, SE [6-VIC NHS ester]	526	543	1 mg	212
6-VIC, SE [6-VIC NHS ester]	526	543	5 mg	213
6-HEX alkyne	533	559	5 mg	241
6-HEX azide	533	559	5 mg	240
6-HEX, SE [6-Carboxy-2',4,4',5',7,7'-hexachlorofluorescein, succinimidyl ester]	533	559	5 mg	202
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]	533	559	100 µmoles	6026
6-HEX phosphoramidite [5'-Hexachlorofluorescein phosphoramidite]	533	559	10x100 µmoles	6024
6-NED alkyne	545	567	1 mg	216
6-NED azide	545	567	1 mg	217
6-NED maleimide	545	567	1 mg	218
6-NED, SE [6-NED NHS ester]	545	567	1 mg	214
6-NED, SE [6-NED NHS ester]	545	567	1 mg	215
Helix Fluor™ 575, succinimidyl ester	553	570	1 mg	251
Tide Fluor™ 3 acid [TF3 acid] Superior replacement for Cy3	546	571	25 mg	2268
Tide Fluor™ 3 alkyne [TF3 alkyne] Superior replacement for Cy3	546	571	1 mg	2255
Tide Fluor™ 3 amine [TF3 amine] Superior replacement for Cy3	546	571	1 mg	2269
Tide Fluor™ 3 azide [TF3 azide] Superior replacement for Cy3	546	571	1 mg	2254
Tide Fluor™ 3 maleimide [TF3 maleimide] Superior replacement for Cy3	546	571	1 mg	2270
Tide Fluor™ 3 succinimidyl ester [TF3 SE] Superior replacement for Cy3	546	571	1 mg	2271
Tide Fluor™ 3 phosphoramidite [TF3 CEP] Superior replacement to Cy3 phosphoramidite	546	571	100 µmoles	2274
Tide Fluor™ 3WS acid [TF3WS acid] Superior replacement for Cy3	551	563	10 mg	2268
Tide Fluor™ 3WS amine [TF3 amine] Superior replacement for Cy3	551	563	1 mg	2347
Tide Fluor™ 3WS maleimide [TF3 maleimide] Superior replacement for Cy3	551	563	1 mg	2344
Tide Fluor™ 3WS succinimidyl ester [TF3WS SE] Superior replacement for Cy3	551	563	1 mg	2346
Tide Fluor™ 4 acid [TF4 acid] Superior replacement for ROX and Texas Red	578	602	10 mg	2285
Tide Fluor™ 4 alkyne [TF4 alkyne] Superior replacement for ROX and Texas Red	578	602	1 mg	2301
Tide Fluor™ 4 amine [TF4 amine] Superior replacement for ROX and Texas Red	578	602	1 mg	2286
Tide Fluor™ 4 azide [TF4 azide] Superior replacement for ROX and Texas Red	578	602	1 mg	2300
Tide Fluor™ 4 maleimide [TF4 maleimide] Superior replacement for ROX and Texas Red	578	602	1 mg	2287
Tide Fluor™ 4, succinimidyl ester [TF4 SE] Superior replacement for ROX and Texas Red	578	602	5 mg	2289
Tide Fluor™ 5WS acid [TF5WS acid] Superior replacement for Cy5	649	664	10 mg	2278
Tide Fluor™ 5WS alkyne [TF5WS alkyne] Superior replacement for Cy5	649	664	1 mg	2276
Tide Fluor™ 5WS amine [TF5WS amine] Superior replacement for Cy5	649	664	1 mg	2279
Tide Fluor™ 5WS azide [TF5WS azide] Superior replacement for Cy5	649	664	1 mg	2275
Tide Fluor™ 5WS maleimide [TF5WS maleimide] Superior replacement for Cy5	649	664	1 mg	2280
Tide Fluor™ 5WS succinimidyl ester [TF5WS SE] Superior replacement for Cy5	649	664	5 mg	2281
Tide Fluor™ 6WS acid [TF6WS acid] Superior replacement for Cy5.5	682	701	10 mg	2291
Tide Fluor™ 6WS alkyne [TF6WS alkyne] Superior replacement for Cy5.5	682	701	1 mg	2303
Tide Fluor™ 6WS amine [TF6WS amine] Superior replacement for Cy5.5	682	701	1 mg	2292
Tide Fluor™ 6WS azide [TF6WS azide] Superior replacement for Cy5.5	682	701	1 mg	2302
Tide Fluor™ 6WS maleimide [TF6WS maleimide] Superior replacement for Cy5.5	682	701	1 mg	2293
Tide Fluor™ 6WS succinimidyl ester [TF6WS SE] Superior replacement for Cy5.5	682	701	1 mg	2294
Tide Fluor™ 7WS acid [TF7WS acid] Superior replacement for Cy7	756	780	10 mg	2330
Tide Fluor™ 7WS alkyne [TF7WS alkyne] Superior replacement for Cy7	756	780	1 mg	2305
Tide Fluor™ 7WS amine [TF7WS amine] Superior replacement for Cy7	756	780	1 mg	2331
Tide Fluor™ 7WS azide [TF7WS azide] Superior replacement for Cy7	756	780	1 mg	2304
Tide Fluor™ 7WS maleimide [TF7WS maleimide] Superior replacement for Cy7	756	780	1 mg	2332
Tide Fluor™ 7WS succinimidyl ester [TF7WS SE] Superior replacement for Cy7	756	780	1 mg	2333
Tide Fluor™ 8WS acid [TF8WS acid] Near Infrared Emission	785	801	10 mg	2335
Tide Fluor™ 8WS alkyne [TF8WS alkyne] Near Infrared Emission	785	801	1 mg	2307
Tide Fluor™ 8WS amine [TF8WS amine] Near Infrared Emission	785	801	1 mg	2336
Tide Fluor™ 8WS azide [TF8WS azide] Near Infrared Emission	785	801	1 mg	2306
Tide Fluor™ 8WS maleimide [TF8WS maleimide] Near Infrared Emission	785	801	1 mg	2337
Tide Fluor™ 8WS succinimidyl ester [TF8WS SE] Near Infrared Emission	785	801	1 mg	2338

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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	454	N/A	5 g	2001
DABCYL C2 amine	454	N/A	100 mg	2006
DABCYL C2 maleimide	454	N/A	25 mg	2008
DABCYL-DBCO	454	N/A	5 mg	2010
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] CAS 146998-31-4	454	N/A	1 g	2004
DABCYL succinimidyl ester [4-((4-(Dimethylamino)phenyl)azo)benzoic acid, succinimidyl ester] CAS 146998-31-4	454	N/A	5 g	2005
3'-DABCYL CPG 1000 Å	454	N/A	1 g	6008
5'-DABCYL C6 Phosphoramidite	454	N/A	1 g	6009
Tide Quencher™ 1 acid [TQ1 acid]	492	N/A	100 mg	2190
Tide Quencher™ 1 alkyne [TQ1 alkyne]	492	N/A	5 mg	2189
Tide Quencher™ 1 amine [TQ1 amine]	492	N/A	5 mg	2192
Tide Quencher™ 1 azide [TQ1 azide]	492	N/A	5 mg	2188
Tide Quencher™ 1 CPG [TQ5 CPG] 500 Å	492	N/A	100 mg	2193
Tide Quencher™ 1 CPG [TQ5 CPG] 1000 Å	492	N/A	100 mg	2194
Tide Quencher™ 1 maleimide [TQ1 maleimide]	492	N/A	5 mg	2196
Tide Quencher™ 1 phosphoramidite [TQ1 phosphoramidite]	492	N/A	100 µmoles	2198
Tide Quencher™ 1 succinimidyl ester [TQ1 SE]	492	N/A	25 mg	2199
Tide Quencher™ 2 acid [TQ2 acid]	516	N/A	100 mg	2200
Tide Quencher™ 2 alkyne [TQ2 alkyne]	516	N/A	5 mg	2212
Tide Quencher™ 2 amine [TQ2 amine]	516	N/A	5 mg	2202
Tide Quencher™ 2 azide [TQ2 azide]	516	N/A	5 mg	2211
Tide Quencher™ 2 CPG [TQ5 CPG] 500 Å	516	N/A	100 mg	2203
Tide Quencher™ 2 CPG [TQ5 CPG] 1000 Å	516	N/A	100 mg	2204
Tide Quencher™ 2 phosphoramidite [TQ2 phosphoramidite]	516	N/A	100 µmoles	2208
Tide Quencher™ 2 succinimidyl ester [TQ2 SE]	516	N/A	25 mg	2210
Tide Quencher™ 2WS acid [TQ2WS acid]	516	N/A	25 mg	2050
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]	516	N/A	1 mg	2213
Tide Quencher™ 2WS alkyne [TQ2WS alkyne]	516	N/A	5 mg	2214
Tide Quencher™ 2WS maleimide [TQ2WS maleimide]	516	N/A	1 mg	2059
Tide Quencher™ 2WS succinimidyl ester [TQ2WS, SE]	516	N/A	5 mg	2058
BXQ-1 Alkyne	522	N/A	1 mg	2414
BXQ-1 Amine	522	N/A	5 mg	2406
BXQ-1 Azide	522	N/A	1 mg	2412
BXQ-1 Carboxylic Acid	522	N/A	25 mg	2400
BXQ-1 CPG (500 Å)	522	N/A	100 mg	2408
BXQ-1 CPG (1000 Å)	522	N/A	100 mg	2410
BXQ-1 Maleimide	522	N/A	1 mg	2404
BXQ-1 Succinimidyl Ester	522	N/A	5 mg	2402
5-TAMRA [5-Carboxytetramethylrhodamine] CAS 91809-66-4	552	578	10 mg	363
5-TAMRA [5-Carboxytetramethylrhodamine] CAS 91809-66-4	552	578	100 mg	364
5-TAMRA [5-Carboxytetramethylrhodamine] CAS 91809-66-4	552	578	1 g	365
Rhodamine aldehyde [5-TAMRA aldehyde]	552	578	5 mg	9005
5-TAMRA alkyne	552	578	5 mg	487
5-TAMRA alkyne	552	578	50 mg	961
5-TAMRA azide	552	578	5 mg	486
5-TAMRA azide	552	578	50 mg	960
5-TAMRA cadaverine	552	578	5 mg	356
5-TAMRA ethylenediamine	552	578	5 mg	358
5-TAMRA C6 maleimide	552	578	5 mg	424
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-68-7	552	578	5 mg	373
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-68-7	552	578	100 mg	374
5-TAMRA, SE [5-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-68-7	552	578	1 g	375
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] CAS 98181-63-6	552	578	100 mg	360
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] CAS 98181-63-6	552	578	1 g	361
5(6)-TAMRA [5(6)-Carboxytetramethylrhodamine] CAS 98181-63-6	552	578	5 g	362
5(6)-TAMRA cadaverine	552	578	25 mg	355
5(6)-TAMRA ethylenediamine	552	578	25 mg	354
5(6)-TAMRA Maleimide [Tetramethylrhodamine-5-(and-6)-maleimide]	552	578	5 mg	412
5(6)-TAMRA C6 maleimide	552	578	5 mg	423
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] CAS 246256-50-8	552	578	25 mg	370
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] CAS 246256-50-8	552	578	100 mg	371
5(6)-TAMRA, SE [5-(and-6)-Carboxytetramethylrhodamine, succinimidyl ester] CAS 246256-50-8	552	578	1 g	372
6-TAMRA [6-Carboxytetramethylrhodamine] CAS 91809-67-5	552	578	10 mg	366
6-TAMRA [6-Carboxytetramethylrhodamine] CAS 91809-67-5	552	578	100 mg	367
6-TAMRA [6-Carboxytetramethylrhodamine] CAS 91809-67-5	552	578	1 g	368
6-TAMRA alkyne	552	578	5 mg	491
6-TAMRA alkyne	552	578	50 mg	966
6-TAMRA azide	552	578	5 mg	490
6-TAMRA azide	552	578	50 mg	965
6-TAMRA cadaverine	552	578	5 mg	357
6-TAMRA CPG 1000 Å	552	578	1 g	6051
6-TAMRA ethylenediamine	552	578	5 mg	359
6-TAMRA Maleimide [Tetramethylrhodamine-6-maleimide] CAS 174568-68-4	552	578	1 mg	419
6-TAMRA C6 maleimide	552	578	5 mg	425
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-69-8	552	578	5 mg	376
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-69-8	552	578	100 mg	377
6-TAMRA, SE [6-Carboxytetramethylrhodamine, succinimidyl ester] CAS#: 150810-69-8	552	578	1 g	378
BXQ-2 Alkyne	554	N/A	1 mg	2434
BXQ-2 Amine	554	N/A	5 mg	2426
BXQ-2 Azide	554	N/A	1 mg	2432
BXQ-2 Carboxylic Acid	554	N/A	25 mg	2420
BXQ-2 CPG (500 Å)	554	N/A	100 mg	2428
BXQ-2 CPG (1000 Å)	554	N/A	100 mg	2430
BXQ-2 Maleimide	554	N/A	1 mg	2424
BXQ-2 Succinimidyl Ester	554	N/A	5 mg	2422
Tide Quencher™ 3 acid [TQ3 acid]	573	N/A	100 mg	2220
Tide Quencher™ 3 alkyne [TQ3 alkyne]	573	N/A	5 mg	2232
Tide Quencher™ 3 amine [TQ3 amine]	573	N/A	5 mg	2222
Tide Quencher™ 3 azide [TQ3 azide]	573	N/A	5 mg	2231
Tide Quencher™ 3 CPG [TQ5 CPG] 500 Å	573	N/A	100 mg	2223
Tide Quencher™ 3 CPG [TQ5 CPG] 1000 Å	573	N/A	100 mg	2224
Tide Quencher™ 3 maleimide [TQ3 maleimide]	573	N/A	5 mg	2226
Tide Quencher™ 3 phosphoramidite [TQ3 phosphoramidite]	573	N/A	100 µmoles	2228
Tide Quencher™ 3 succinimidyl ester [TQ3 SE]	573	N/A	25 mg	2230
Tide Quencher™ 3WS acid [TQ3WS acid]	573	N/A	1 mg	2229
Tide Quencher™ 3WS succinimidyl ester [TQ3WS SE]	573	N/A	5 mg	2227
Tide Quencher™ 4 CPG [TQ5 CPG] 500 Å	603	N/A	100 mg	2062
Tide Quencher™ 4 CPG [TQ5 CPG] 1000 Å	603	N/A	100 mg	2063
Tide Quencher™ 4WS acid [TQ4WS acid]	603	N/A	5 mg	2060
Tide Quencher™ 4WS alkyne [TQ4WS alkyne]	603	N/A	1 mg	2069
Tide Quencher™ 4WS amine [TQ4WS amine]	603	N/A	1 mg	2061
Tide Quencher™ 4WS azide [TQ4WS azide]	603	N/A	1 mg	2068
Tide Quencher™ 4WS maleimide [TQ4WS maleimide]	603	N/A	1 mg	2064
Tide Quencher™ 4WS succinimidyl ester [TQ4WS SE]	603	N/A	1 mg	2067
Tide Quencher™ 5 CPG [TQ5 CPG] 500 Å	661	N/A	100 mg	2077
Tide Quencher™ 5 CPG [TQ5 CPG] 1000 Å	661	N/A	100 mg	2078
Tide Quencher™ 5WS acid [TQ5WS acid]	661	N/A	5 mg	2075
Tide Quencher™ 5WS alkyne [TQ5WS alkyne]	661	N/A	1 mg	2083
Tide Quencher™ 5WS amine [TQ5WS amine]	661	N/A	1 mg	2076
Tide Quencher™ 5WS azide [TQ5WS azide]	661	N/A	1 mg	2082
Tide Quencher™ 5WS maleimide [TQ5WS maleimide]	661	N/A	1 mg	2079
Tide Quencher™ 5WS succinimidyl ester [TQ5WS SE]	661	N/A	1 mg	2081
Tide Quencher™ 6WS acid [TQ6WS acid]	694	N/A	5 mg	2090
Tide Quencher™ 6WS alkyne [TQ6WS alkyne]	694	N/A	1 mg	2098
Tide Quencher™ 6WS amine [TQ6WS amine]	694	N/A	1 mg	2091
Tide Quencher™ 6WS azide [TQ6WS azide]	694	N/A	1 mg	2097
Tide Quencher™ 6WS maleimide [TQ6WS maleimide]	694	N/A	1 mg	2094
Tide Quencher™ 6WS succinimidyl ester [TQ6WS SE]	694	N/A	1 mg	2096
Tide Quencher™ 7WS acid [TQ7WS acid]	764	N/A	5 mg	2105
Tide Quencher™ 7WS alkyne [TQ7WS alkyne]	764	N/A	1 mg	2113
Tide Quencher™ 7WS amine [TQ7WS amine]	764	N/A	1 mg	2106
Tide Quencher™ 7WS azide [TQ7WS azide]	764	N/A	1 mg	2112
Tide Quencher™ 7WS maleimide [TQ7WS maleimide]	764	N/A	1 mg	2109
Tide Quencher™ 7WS succinimidyl ester [TQ7WS SE]	764	N/A	1 mg	2111

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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	-	-	-	-	-	-	+	+++

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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™ 1	345	442	20000	0.95	0.246	0.187
Tide Fluor™ 2WS	502	525	75000	0.9	0.211	0.091
Tide Fluor™ 2	500	527	75000	0.9	0.288	0.201
Tide Fluor™ 3WS	555	565	150000	0.105	0.079	0.079
Tide Fluor™ 3	555	584	85000	0.85	0.331	0.201
Tide Fluor™ 4	590	618	90000	0.91	0.489	0.436
Tide Fluor™ 5WS	649	664	250000	0.25	0.023	0.027
Tide Fluor™ 6WS	676	695	220000	0.18	0.111	0.009
Tide Fluor™ 7WS	749	775	275000	0.12	0.009	0.049
Tide Fluor™ 8WS	775	807	250000	0.08	0.103	0.109

ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
Φ = Fluorescence quantum yield in aqueous buffer (pH 7.2).
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).
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

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™ 1	488	20,000	0.147	0.194
Tide Quencher™ 2	512	21,000	0.100	0.120
Tide Quencher™ 2WS	515	21,000	0.100	0.120
Tide Quencher™ 3	576	22,000	0.085	0.091
Tide Quencher™ 3WS	576	90,000	0.186	0.205
Tide Quencher™ 4	604	23,000	0.146	0.183
Tide Quencher™ 4WS	604	90,000	0.149	0.136
Tide Quencher™ 5	661	24,000	0.170	0.082
Tide Quencher™ 5WS	661	130,000	0.072	0.082
Tide Quencher™ 6WS	691	130,000	0.120	0.102
Tide Quencher™ 7WS	759	140,000	0.072	0.091

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ε = molar extinction coefficient at their maximum absorption wavelength (Units = cm^-1M^-1).
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).
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).

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