digital PCR

Summary

Digital PCR (digital PCR) is to convert the exponential multiplication signal of traditional PCR into a linear digital signal, which can be read by specific instruments and analyzed statistically to analyze the PCR products. Currently, there is one main method used for digital PCR: general digital PCR.

Principle

The basic principle of digital PCR is to divide a sample into hundreds or thousands of copies so that each reaction bath contains as few copies of the target molecule (i.e., the template) as possible, then add fluorescently labeled probes and DNA polymerase to each bath to amplify the DNA samples in batch, and then read the fluorescence signals to reflect the PCR amplification of the target molecules.

The advent of digital PCR has significantly improved the efficiency of life science research, allowing biologists to rapidly and in high throughput detect many changes in disease-causing genes in patient samples, including mutations, identification of allelic imbalances, and more.

Appliance

Common application areas for digital PCR are as follows: detection of locus mutations and allelic imbalances in tumor tissues or DNA samples.

Operation method

Plain digital PCR

Principle

The basic principle of digital PCR is that the analyzed sample DNA is diluted to an average of approximately one sample molecule per two wells and then equally divided into multiwell plates. A single copy of the template is amplified using PCR under optimal conditions. The amplified product is hybridized to a molecular beacon (MB) fluorescent probe and sequence-specific PCR products are detected based on different fluorescence. Digital PCR directly calculates the two-by-two comparison. Digital PCR directly calculates the number of alleles per sample in a two-by-two comparison (e.g., sire versus dam, or wild type versus mutant). At the same time, statistical analyses can be used to analyze the reliability of the differences between two samples, such as Bayesian-type likelihood methods. Molecular beacon probe working principle: M is a stem-loop double-labeled oligonucleotide probe with a hairpin structure, which has a fluorescent group attached at the 5' end and a burst group (Dabcyl group) attached at the 3' end. The nucleic acid sequences of the two ends are complementary paired, so the burst group at the 3' end is close to the fluorescent group at the 5' end, and the photon generated by the fluorescent group after excitation is burned out by the burst group. When the MB primer is paired with the template, the 5' and 3' ends of the MB primer are separated, and the molecular beacon becomes a chain rather than a hairpin, separating the fluorescent group from the bursting group and relieving the bursting effect. Energy is generated when the fluorescent group is excited and released as infrared light waves.

Materials and Instruments

Equipment:
PCR instrument, centrifuge, fluorescence spectrophotometer or dual-channel fluorescence quantitative PCR instrument, UV spectrophotometer.
Reagents:
(1) Buffers and solutions: 67 mmol/L Tris (pH 8.8).
16.6 mmol/L (NH4)
2
SO
4
(NH4) 2 SO 4, 6.7 mmol/L MgCl
2
MgCl2, MgCl2, MgCl2
10 mmol/L β-mercaptoethanol, 20 mmol/L dATP, 20 mmol/L dCTP, 20 mmol/L dGTP, 20 mmol/L dTTP, and 6% DMSO.
(2) Enzyme Taq polymerase;
(3) Nucleic acids and oligonucleotides:
① Exogenous or target DNA;
Primers and probes: 1 μmol/L primer F1, 1 μmol/L primer R1, 5 μmol/L primer INT, 1 μmol/L green fluorescent molecular beacon (MB-green), 1 μmol/L red fluorescent molecular beacon (MB-red), and the primers were dissolved in 50 μmol/L TE buffer.

Move

The basic process of digital PCR can be divided into the following steps:

(i) Primer design

Primers F1 and R1 are two specific primers used to amplify the target fragment; primer INT is used to generate a single-stranded target fragment in PCR amplification; MB-red is an MB probe used to detect PCR products from any segment of the genome other than the target fragment, including wild-type and mutant; and MB-green is an MB probe for detecting PCR products from the wild-type target fragment. MB-green is an MB probe that detects PCR products from a wild-type target fragment.

Mutations within the amplified target fragment significantly prevent hybridization of the PCR product to the MB-green probe, and a comparison of the fluorescence signal intensity of MB-green/MB-red gives an indication of the proportion of wild-type DNA in the total sample, and hence the proportion of mutant DNA in the total sample.

(ii) PCR amplification reaction

A. Dilute genomic DNA samples extracted from tissues or body fluids in a gradient and add them to a 96-well PCR plate, containing approximately 0.5 genomic DNA per well.

Note: Diluted DNA samples need to be assayed using a conventional PCR reaction to determine the addition of approximately 0.5 genomic DNA per well. the amount of template per well is approximately 1.5 pg, which is defined as 0.5 template molecules per well.

B. Make up 700 μl of working solution according to the following ratios, then add 7 μl of working solution to each well of the 96-well PCR plate, mix well and perform the PCR reaction.

PCR working solution ratio:
10× amplification buffer 70 μl
20 mmol/L 4 dNTP mixture (pH8.0) 35 μl
20 μmol/L forward primer F1 35 μl
20 μmol/L reverse primer R1 35 μl
1~5 U/μl Platinum ○RTaq DNA polymerase 35 U
_H2O 490-518 μl
total volume 700 μl

Standard PCR reaction conditions.

Reagents Concentration
MgCl2 6.7 mmol/L
Tris (pH 8.8) 67 mmol/L
(NH4)2SO4 16.6 mmol/L
β-mercaptoethanol 10 mmol/L
dNTPs 1 mmol/L
DMSO 6% (v/v)
Primers 1 μmol/L
Platinum○RTaq DNA polymerase 0.05 U/μL
Template DNA 0.5 molecules

C. Add a drop of light mineral oil (~50 μl) to the top of the reaction mixture to prevent evaporation of the sample during multiple cycles of the PCR reaction.

D. Place the 96-well PCR plate on the PCR instrument. Perform PCR amplification as follows. as follows: 94 ℃, 1 min; 94 ℃, 15 s; 55 ℃, 15 s for 60 cycles; 72 ℃, 15 s; 70 ℃, 5 min.

Note: The reaction system was subjected to fluorescence analysis immediately or stored at room temperature for a maximum of 36 h, and then subjected to fluorescence analysis.

(iii) Fluorescence analysis

A. Prepare 400 μl of working solution according to the following components, and add 3.5 μl of working solution to each well of the 96-well plate:

10× Amplification Buffer 40 μl
20 mmol/L 4 kinds of dNTP mixture (pH 8.0) 20 μl
20 μmol/L Forward Primer INF 100 μl
20 μmo/L MB-green 20 μl
20 μmo/L MB-red 20 μl
1~5 U/μl Platinum ○RTaq DNA Polymerase 40 U
H2O 160-192 μl
Total volume 400 μl

Standard digital PCR reaction conditions:
Reagent concentration
MgCl₂ 6.7 mmol/L
Tris (pH 8.8) 67 mmol/L
(NH4)2SO4 16.6 mmol/L
β-mercaptoethanol 10 mmol/L
dNTPs 1 mmol/L
DMSO 6% v/v/v
Primer INT 5 μmol/L
MB-green 1 μ mol/L
MB-red 1 μmol/L
Platinum○RTaq DNA polymerase 0.05U/μL

B. Centrifuge the 96-well PCR plate at 6000 g for 20 s.
C. Place the 96-well PCR plate into the PCR instrument and perform PCR amplification as follows. The corresponding cycling conditions and temperatures are as follows:
94°C, 1min
94°C, 15s
55°C, 15s Perform 10-15 cycles
72°C, 15s
94°C, 1min
60°C, 5min

D. Incubate the PCR plate at room temperature for 10-60 min, and then place it in a fluorescence spectrophotometer to excite MB-green fluorescence at 485 nm/530 nm and MB-red at 530 nm/590 nm. Calculate the red/green ratio = MB-red fluorescence intensity/MB-green fluorescence intensity, and correct the red/green ratio by using a positive control. Calculate: red/green ratio = MB-red fluorescence intensity/MB-green fluorescence intensity and use a positive control to correct the red/green ratio.

Caveat

① Nested PCR cannot be used during digital PCR amplification because it is not only inconvenient but also leads to contamination problems if it is used during digital PCR.① Nested PCR cannot be used during digital PCR because it is inconvenient and can lead to contamination problems if used during digital PCR.③ The digital PCR reaction does not take a long time (about 2.5 h), but it requires more cycles than normal PCR, much more than the number of cycles normally used. This is because in some wells the template may take several cycles before the amplification reaction starts. The high number of cycles ensures that as long as there is template in the wells, almost equal PCR products are produced in each PCR well.MB-green fluorescence was detected by excitation/emission at 485 nm/530 nm and MB-red fluorescence was detected by excitation/emission at 530 nm/590 nm. Fluorescence is typically in the range of 10,000-20,000 specific fluorescence units (SFU) per well without the addition of DNA template molecules.

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