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AAT Bioquest

Polymerase Chain Reaction (PCR)

PCR (polymerase chain reaction) is a core technique used extensively in molecular biology research to amplify a specific DNA template in vitro rapidly. It enables researchers to generate significant quantities of sample DNA for a wide range of downstream laboratory and clinical applications, including cloning, genotyping, sequencing, mutagenesis, forensics, and the detection of pathogens to diagnose infectious diseases. Since being introduced in 1985, several iterations of the PCR process have been developed, including quantitative PCR (qPCR) for monitoring DNA amplification in real-time and reverse-transcription PCR (RT-PCR) for the detection of RNA, a tool that has become instrumental in viral diagnostics.

For accurate and sensitive PCR detection, AAT Bioquest offers a comprehensive portfolio of PCR reference dyes, deoxynucleotide triphosphates, double-stranded DNA-binding dyes, and fluorescent reporter dyes and non-fluorescent quenchers for the development of sequence-specific molecular beacons.

 

 

Principles of PCR


PCR is commonly used to amplify a single DNA template into millions or billions of identical copies in vitro. A typical amplification reaction requires a DNA template, thermostable DNA polymerase, forward and reverse primers, deoxynucleotide triphosphates (dNTPs), and a reaction buffer (Table 1). The components are combined in a PCR or Eppendorf tube and then placed in a thermal cycler to facilitate the amplification process. While inside the thermal cycler, the PCR mixture undergoes a series of temperature and time adjustments which includes three key steps: (1) template denaturation, (2) primer annealing, and (3) primer extension.

PCR Process


During the denaturation process, the double-stranded DNA template is heated at 95°C for two minutes. The high temperature causes hydrogen bonds between the complementary base pairs in the DNA template to separate into two single-stranded components. Next, the temperature is reduced to 50-65°C for approximately 20-40 seconds to facilitate primer annealing to each single-stranded DNA. After annealing, the temperature is increased to 70-74°C to initiate elongation. In this step, DNA polymerases will bind to and extend the primer to form a nascent DNA strand. It does so by moving along the DNA template base by base in the 5' to 3' direction and adds the corresponding complementary dNTP from the reaction mixture. Altogether, these three steps are referred to as a PCR cycle, and after each cycle, the number of double-stranded DNA fragments doubles. PCR cycles are repeated 25 to 30 times to amplify the original DNA template exponentially.


Video 1. PCR animation. Animated video tutorial illustrating the three key steps in the polymerase chain reaction: (1) template denaturation, (2) primer annealing, and (3) primer extension.

 

Table 1. Summary of the components required for PCR

PCR Component
Function
DNA Template This is the sample DNA that contains the target sequence to be amplified.
Thermostable DNA polymeraseThe enzyme that catalyzes the formation of new DNA strands complementary to the target sequence. Commonly used polymerases include TaqDNA polymerase and PfuDNA polymerase.
Primers (forward and reverse)Short, single-stranded DNA sequences that hybridize to the sample DNA and start the process of replication. Primers are designed to complement the sequences at the beginning and end of the DNA template intended for amplifying.
Deoxynucleotide triphosphates (dNTPs)These are the "building blocks" from which the DNA fragments will be synthesized and are commonly available in a ready-to-use format, such as ReadiUse™ dNTP Mix *10 mM* (Cat No. 17200)
Reaction buffer containing magnesiumThis provides a stable environment for the PCR reaction. It has a suitable pH of 8.0 to 9.5 and is fortified with magnesium chloride, a co-factor of DNA polymerase.


Conventional Analysis of PCR Products


Once the PCR process is complete, the reaction products are stained with ethidium bromide (EtBr) and analyzed by agarose gel electrophoresis to determine the size and concentration of the DNA molecules. While ethidium bromide is the most commonly used dye for visualizing DNA, it is mutagenic and highly toxic through inhalation. Therefore, consider using non-toxic alternatives such as Helixyte™ Green (Cat No. 17590), Helixyte™ Gold (Cat No. 17595), Gelite™ Green (Cat No. 17589), or Gelite™ Orange (Cat No. 17594).

 

Table 2. Non-toxic DNA stains for agarose gel electrophoresis

Product
Ex (nm)
Em (nm)
Unit Size
Cat No.
Gelite™ Safe DNA Gel Stain *10,000X Water Solution*513 nm 552 nm 1 mL 17702
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* 513 nm 552 nm 1 mL 17706
Gelite™ Green Nucleic Acid Gel Staining Kit 254 or 300 nm1Long path green filter21 Kit17589
Gelite™ Orange Nucleic Acid Gel Staining Kit 254 or 300 nm1Long path green filter21 Kit17594
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* 497 nm521 nm1 mL17590
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* 497 nm521 nm100 µL17604
Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution* 496 nm539 nm1 mL17595
  1. Excitation settings are for a transilluminator or laser-based gel scanner.
  2. Common long path green filters include the SYBR® filter and GelStar® filter.

 

Types of PCR


Conventional PCR methods, like the process aforementioned, can only amplify DNA and requires agarose gel electrophoresis to determine PCR success from the end-point of the reaction. This process is very time-consuming and is hindered by various caveats, including low sensitivity, low resolution, poor precision, non-automation, post-PCR processing, and a short dynamic range. To address these concerns, several iterations of the PCR process have been developed, including quantitative PCR (qPCR) for monitoring DNA amplification in real-time and reverse-transcription PCR (RT-PCR) for the detection of RNA, a tool that has become instrumental in viral diagnostics.

Reverse-Transcription 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 reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The cDNA is then used as a template for exponential amplification using standard PCR procedure (denaturation, annealing, and elongation). RT-PCR is used in various applications, including gene expression analysis, microarray validation, pathogen detection, and disease research.

Quantitative PCR


Quantitative or real-time PCR (qPCR) enables researchers to monitor the amplification of a DNA template in real-time and not at its end-point, as in conventional PCR. It does so using fluorescent reporter molecules which bind to and detect products generated during each cycle of the PCR process. As the reaction proceeds, fluorescence increases due to the accumulation of the PCR product with each amplification cycle. These fluorescent reporter molecules include dyes that bind to the double-stranded DNA (dsDNA), such as Helixyte™ Green (Cat No. 17591) and Q4ever™ Green (Cat No. 17608), or fluorescently labeled sequence-specific probes, such as TaqMan® probes, Molecular Beacons and Scorpion® probes.

Dye-Based qPCR


In qPCR, dsDNA binding dyes are frequently used as fluorescent reporters to measure gene expression. The fluorescence of the reporter dye increases as the product accumulates with each successive cycle of amplification. Recording the amount of fluorescence emission at each cycle makes it possible to monitor the PCR reaction during the exponential phase. Compared to microarrays, qPCR is more sensitive at detecting modest changes in expression levels, making it well-suited for investigating small subsets of genes. Although dsDNA-binding dyes provide the most convenient and cheapest option for qPCR, the principal drawback to intercalation-based detection of PCR product accumulation is that both specific and nonspecific products generate signals.

qPCR with Cyber Green

qPCR using Helixyte™ Green (Cat No. 17591). During the extension phase, DNA polymerase extends the sequence-specific primer by incorporating dNTPs complementary to the DNA template. As newly synthesized double-stranded DNA is produced, Helixyte™ Green will bind to the DNA complexes and fluoresce (figure made in BioRender).

 

Table 3. 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


Probe-Based qPCR


Probe-based qPCR utilizes target-specific probes to precisely measure DNA amplification at each cycle of the PCR reaction. While probe designs may vary, they typically share three key elements: a short oligonucleotide that is complementary to the target sequence, a fluorescent reporter dye-labeled to the 5' end, and a quencher dye on the 3' end (for information on recommended FRET pairs, see table below). Due to the biochemical phenomenon known as Förster resonance energy transfer (FRET), the fluorescence of the reporter dye is masked by the quencher dye while the probe remains intact. As DNA polymerase extends the primer during elongation, it also hydrolyzes sequence-specific probes that have annealed to the single-stranded DNA template, separating the reporter dye from the quencher and resulting in an amplification-dependent increase in fluorescence. Probe-based qPCR is a favorable method beneficial for specific hybridization, no false positives, and multiplex analysis of multiple target sequences in a single reaction tube.

Probe-based qPCR

Illustration of probe-based qPCR. As DNA polymerase extends the primer during elongation, it hydrolyzes sequence-specific probes that have annealed to the single-stranded DNA template, separating the reporter dye from the quencher and resulting in an amplification-dependent increase in fluorescence (figure made in BioRender).

 

Table 4. 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 5. 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

 

Additional Resources



Recommended FRET Pairs

 

Table 6. 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
------++++


ROX Reference and Reporter Dye Combinations

 

Table 7. Possible ROX Reference and reporter dye combinations for multiplex qPCR assays.

Instrument
Reference Dye
Reporter Dye 1
Reporter Dye 2
Reporter Dye 3
Reporter Dye 4
ABI PRISM® 7700ROX6-FAM6-TET--
ABI PRISM® 7000 and 7900
Applied Biosystems® 7300
StepOnePlus™
ROX6-FAM6-TET6-HEX-
Applied Biosystems® 7500ROX6-FAM6-TET6-HEXTide Fluor™ 3
iFluor® 647
Alexa Fluor 647
Cy5