What is the difference between Real-time PCR, traditional PCR and digital PCR?

Question

Accepted Answer

Basis of Differentiation
Real-time PCR
Traditional PCR
Digital PCR

Definition
Is a PCR-based technique that combines amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction
Is a laboratory technique used to amplify a specific DNA segment using a small amount of starting material such as a DNA template or target sequence 
Is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample

Detection/ measurement of PCR amplification
Measures PCR amplification as it happens in real time throughout the reaction
Measures the amount of accumulated PCR product at the end of the PCR cycles
Measures the number of target molecules directly by counting positive fluorescence in compartments

Type of process
Automated
Non-automated
Can be easily automated for higher precision

Type of reaction
Is a quantitative reaction &ndash; data is collected during the exponential growth phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid
Is a semi-quantitative reaction &ndash; comparing the intensity of the amplified band on a gel to standards of a known concentration can provide 'semi-quantitative' results
Is a quantitative reaction - allows absolute quantitation of the target molecule using a unique blend of sample dilution and Poisson statistical algorithm

Sensitivity & Accuracy
Very high sensitivity and accuracy
Low sensitivity and accuracy
Desired level of accuracy can be achieved by increasing the number of replicates

Advantages

- Collects data in the exponential growth phase of PCR for higher accuracy
- Increased dynamic range of detection
- Detection is capable down to a 2-fold change
- The cleaved probe provides a permanent record amplification of an amplicon

- Does not offer any advantages over Real-time PCR or Digital PCR

- Capable of analyzing complex mixtures
- More tolerant to PCR inhibitors
- No need to rely on standards or references
- Capable of detecting small fold change differences because of its linear response to the number of copies present

Basis of Differentiation	Real-time PCR	Traditional PCR	Digital PCR
Definition	Is a PCR-based technique that combines amplification of a target DNA sequence with quantification of the concentration of that DNA species in the reaction	Is a laboratory technique used to amplify a specific DNA segment using a small amount of starting material such as a DNA template or target sequence	Is a quantitative PCR method that provides a sensitive and reproducible way of measuring the amount of DNA or RNA present in a sample
Detection/ measurement of PCR amplification	Measures PCR amplification as it happens in real time throughout the reaction	Measures the amount of accumulated PCR product at the end of the PCR cycles	Measures the number of target molecules directly by counting positive fluorescence in compartments
Type of process	Automated	Non-automated	Can be easily automated for higher precision
Type of reaction	Is a quantitative reaction – data is collected during the exponential growth phase of PCR when the quantity of the PCR product is directly proportional to the amount of template nucleic acid	Is a semi-quantitative reaction – comparing the intensity of the amplified band on a gel to standards of a known concentration can provide 'semi-quantitative' results	Is a quantitative reaction - allows absolute quantitation of the target molecule using a unique blend of sample dilution and Poisson statistical algorithm
Sensitivity & Accuracy	Very high sensitivity and accuracy	Low sensitivity and accuracy	Desired level of accuracy can be achieved by increasing the number of replicates
Advantages	- Collects data in the exponential growth phase of PCR for higher accuracy - Increased dynamic range of detection - Detection is capable down to a 2-fold change - The cleaved probe provides a permanent record amplification of an amplicon	- Does not offer any advantages over Real-time PCR or Digital PCR	- Capable of analyzing complex mixtures - More tolerant to PCR inhibitors - No need to rely on standards or references - Capable of detecting small fold change differences because of its linear response to the number of copies present

What is the difference between Real-time PCR, traditional PCR and digital PCR?

Posted March 25, 2022