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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 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.


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

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

 

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
----+++++-


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