Quantitative PCR

Summary

Quantitative PCR is a technique to quantify the amount of PCR starting template by analyzing the PCR end product or monitoring the PCR process using a standard as a control.

According to the different principles, the main quantitative PCR methods used at present include limiting dilution method, quantitative PCR with internal reference, fluorescence quantity PCR (FQ-PCR), etc. Among them, fluorescence quantity PCR is the newest method, which can be used to quantify the amount of PCR template.

Among them, FQ-PCR is a newly developed quantitative PCR technique.

Principle

The basic principle of fluorescence quantitative PCR is to add fluorescent groups in the PCR reaction system, and continuously detect the change of fluorescent signal in the reaction system during the PCR reaction, when the signal is enhanced to a certain threshold, the number of cycles at this time, i.e., the cycle threshold Ct (cycle threshold, Ct), is recorded.

There is a strict linear relationship between the cycle parameter Ct and the logarithm of the number of starting templates in the PCR system, and a standard curve is made by plotting the Ct values amplified using positive quantitative standard templates of different gradients and the number of templates of the positive quantitative standard after logarithmic fitting.

Finally, according to the Ct value of the sample to be tested, the number of starting templates can be accurately determined from the standard curve. Depending on the probe used, there are two types, Taq-man fluorescent probes and fluorescent dyes.

Taqman works by adding a fluorescently labeled probe to the PCR system, which specifically hybridizes with the primer-amplified product DNA template. The 5' end of the probe is labeled with the fluorescent emitting group FAM (peak fluorescence emission at 518 nm), and the 3' end is labeled with the fluorescent bursting group TAMRA (6-carboxytetramethylrhodamine, peak fluorescence emission at 582 nm). ).

The terminal base is phosphorylated to prevent the probe from being extended during PCR amplification. When the probe remains intact, the bursting group inhibits the fluorescence emission of the emitting group, and once the emitting group separates from the bursting group, the inhibition is lifted and the increase in optical density at 518 nm is detected by the fluorescence system.

During the replication process of PCR, the probe hybridizes with the template DNA, and during the extension process, Taq enzyme extends with the primer to the position where the probe binds to the template, exerting the exonuclease activity of 5' → 3' to cut off the probe, and the fluorescent signal of FAM is released.

In this way, for every 1 replication of the template, 1 probe is cut off, accompanied by the release of 1 fluorescent signal.

There is a one-to-one relationship between the fluorescent signal intensity and the amount of PCR product, so this technique allows dynamic observation of the PCR reaction process and accurate quantification of the template.

SYBR is an asymmetric cyanine fluorescein that is non-specifically embedded in the minor grooves in the DNA double helix structure and emits a fluorescent signal, whereas Sybr dye molecules that are not doped into the strand do not emit any fluorescent signals, thus guaranteeing that the increase in fluorescent signals is completely synchronized with the increase in PCR products.

This method avoids the need for designing and labeling fluorescent probes and the use of expensive and complex reagents, and is suitable for any PCR amplification system.

Operation method

fluorescence quantitative PCR

Principle

The basic principle of fluorescence quantitative PCR is to add fluorescent groups to the PCR reaction system, and continuously detect the change of fluorescence signal in the reaction system during the PCR reaction process. When the signal is enhanced to a certain threshold, the number of cycles, i.e., the cycle threshold (Ct), is recorded. There is a strict linear relationship between the cycle parameter Ct and the logarithm of the number of starting templates in the PCR system. The Ct values amplified by different gradients of positive quantitative standard templates and the number of templates of the positive quantitative standard are plotted by logarithmic fitting to make a standard curve. Finally, according to the Ct value of the sample to be tested, the number of starting templates can be accurately determined from the standard curve. According to the different probes used, there are two kinds, one is Taq-man fluorescent probe and the other is fluorescent dye. SYBR is an asymmetric cyanine fluorescein, which is non-specifically embedded in the groove in the double helix structure of the DNA, emitting fluorescent signals, while Sybr dye molecules that are not doped into the strand do not emit any fluorescent signals, thus ensuring that the increase in fluorescence signals is completely synchronized with the increase in the PCR product. increase of the fluorescence signal is completely synchronized with the increase of the PCR product. This method eliminates the need for designing and labeling fluorescent probes and the use of expensive, complex reagents and is suitable for any PCR amplification system.

Materials and Instruments

Equipment:
① fluorescence quantitative PCR instrument
② Ultraviolet spectrophotometer
③ Micro pipette (20 μl)
④ 0.2 ml Ep tube (sterilized)
Reagents:
①Materials: standard DNA template, sample DNA
②SYBR I fluorescent dye
①SYBR I fluorescent dye ③Upstream and downstream primers
③ Upstream and downstream primers ④ dNTP mixture
⑤ Taq DNA polymerase
⑥10 x PCR reaction buffer
⑦ Sterilized water

Move

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

(i) Reagent preparation

(1) Upstream primers and downstream primers Concentration 20 μmol/L.

(2) dNTP mixture 100 mg of dATP, dGTP, dCTP and dTTP sodium salt, add 2 ml of deionized water to dissolve, adjust the pH value to 7.0-7.5 with 0.1 mol/L NaOH to make the concentration of 5 mmol/L, and store at -20 ℃ after loading.

Commercialized mixture (2 mmol/L each) is also available.

(3) Taq DNA polymerase 5 U/μl, with a final concentration of 1-2.5 U in a 50 μl reaction volume.

(4) PCR reaction buffer (10x) 500 mmol/L KCI, 100 mmol/L Tris-HC1 (pH 8.4), 15 mmol/L MgCl₂.

(ii) Preparation of quantitative standards

(1) Add 1 μg of standard DNA template, 2 μl each of specific upstream and downstream primers, 5 μl of 10 x PCR buffer, 5 U of Taq DNA polymerase, 50 μl of sterilized water, and mix well in an Ep tube.

(2) The amplification reaction was performed on a PCR instrument: denaturation at 95 ℃ for 30 s, annealing at 55 ℃ for 30 s, extension at 72 ℃ for 1 min for 30 cycles.

(3) The PCR products were purified, sequenced and verified, OD values were determined, and the number of copies per milliliter was converted to molecular mass and stored.

(iii) Preparation of quantitative standard curve

(1) Dilute the quantitative standards ¹⁰², ¹⁰⁴, ¹⁰⁶, ¹⁰⁸ and ¹⁰¹⁰ times, take 5 μl of the diluted standards and add the PCR reaction reagents as described above, plus 2 μl of Sybr I fluorescent dye, for a total volume of 50 μl.

(2) Carry out the PCR reaction on the fluorescence quantitative PCR instrument according to the above PCR reaction conditions, and generate the quantitative standard curve automatically by computer.

(D) Sample Determination

(1) Add 5 μl of sample DNA, 2 μl each of upstream and downstream primers, 5 μl of 10 x PCR buffer, 5 μl of Taq DNA polymerase, 2 μl of SYBR I fluorescent dye, 50 μl of sterilized water to the Ep tube and mix well.

The amplification reaction was carried out on a fluorescence quantitative PCR instrument according to the above PCR reaction conditions.

(2) According to the results of the sample measurement, combined with the quantitative standard curve, the computer self-calculates the copy number in the sample.

Caveat

(1) Strictly optimize the reagents and conditions for PCR amplification reaction.(1) The template should be prepared in such a way as to avoid the degradation of DNA, and the DNA should be purified as much as possible so that it does not contain any protease, nuclease, Taq DNA polymerase inhibitor and proteins that can bind to DNA.In order to ensure the specific amplification of PCR, the design of primers should comply with the following principles: the length of primers should be 15-30 bases, and the difference in the length of upstream and downstream primers should not be more than 3 bases.（The content of (G+C) should be 40%-60%, and the four bases should be evenly distributed in the primers; there should be no complementary sequences in the primers themselves, and there should be no inverse repeating sequences larger than 3 bp, otherwise the primers will fold and form a hairpin structure.There should not be more than 4 complementary or homologous bases between the upstream and downstream primers, and the overlap of complementary at the 3' end should be avoided to prevent the formation of primer dimer.The 3' end of the primer should not be modified in any way, nor should there be any possibility of secondary structure formation, and T should not be used for the 3' end as much as possible.The 5' end of the primer can be modified, plus some useful but not complementary to the target DNA sequence calculated from the two primers can not be more than 5 ℃ difference in the unlinking temperature.(iii) Primer concentration should be determined by pre-test to determine the optimal concentration, the concentration range is generally 0.2-1.0 μmol / L. (iv) Cycling parameters in PCR to control the optimal concentration of primers.The control of temperature in PCR is an important part of the success or failure of the experiment. The annealing temperature and time depend on the length and concentration of the primer and the content of (G+C) in the base composition. Generally speaking, the annealing temperature is the unlinking temperature of the primer minus 5 ℃.The extension temperature depends on the optimal temperature of Taq DNA polymerase, usually 70-75 ℃, and the extension time depends on the length of the amplified fragments.⑤PCR buffer MgMg+The concentration of Mg 2+ has a significant effect on the specificity and yield of the PCR products, and the concentration of various mononucleotides is 200 μmol/L. The Mg 2+ buffer is used as the buffer for the PCR products.Mg2+A concentration of 200 μmol/L for all mononucleotides and 1.5 mmol/L for Mg 2+ was suitable.In addition, different manufacturers of Taq DNA polymerase have different buffer compositions, so care should be taken to ensure that they are compatible.(6) To avoid non-specific amplification, the "hot start method" of Taq DNA polymerase can be used.(2) Setting up control experiments: In order to ensure the certainty of the results, a positive control and a negative control or blank control should be set up.(3) Prevent PCR contamination.(4) Repeat the experiment for the same set of reference standards with multiple independently prepared samples, and the obtained experimental results are analyzed for statistical significance to standardize the experimental method and make it reproducible.

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