Establishment of a real-time PCR quantitative analysis experiment

Summary

The process of establishing a quantitative real-time PCR assay for a new target molecule involves: (i) a design step for the selection of PCR primers and probes; and (ii) the selection of the real-time PCR assay method according to the requirements of the particular assay. Real-time PCR using SYBRGreen is often used to optimize primers and is primarily used in research to detect PCR products TaqMari probes and molecular signals provide better specificity. This experiment was derived from PCR Laboratory Guide (Second Edition) by Kang Seed and Lijia Qu.

Operation method

Establishment of a real-time PCR quantitative analysis experiment

Materials and Instruments

MgCl2 probe HotStmMix forward and reverse amplification primers Template DNA
PCR instrument Reactor tubes, capillaries or 96-well microtitre plates

Move

I. Materials

1. Buffers and solutions

MgCl₂ (25 mmol/L)

Probes (e.g. Molecular Signals) or SYBRGreen (MolecularProbes)

2. Enzymes and enzyme buffers

HotStmMix, 2X (contains TaqDNA polymerase, PCR buffer and dNTP)

3. nucleic acids and oligonucleotides

Forward and reverse amplification primers

Template DNA

4. Specialized equipment

Real-time PCR instrument

Suitable reaction tubes, capillary tubes or 96-well microtitre plates (chosen according to the different methods)

II. METHODS A----ABI7700ORI-CYCLER

1. Set up the following PCR components for each reaction on a 96-well microtitre plate. Mix the HotStart mixture with primers and probes/SYBRGreen and add the template in the required amount. There are five 10-fold dilutions of template DNA.

2X HotStartMix 25ul
Primer A 0.1~ 0.5umol/L
Primer B 0.1~ 0.5umol/L
Probe or SYBRGreen 0.1~ 0.5umol/L
MgCl2(25 mmol/L) 3~6 mmol/L
Template DNA (500ng~50pg) 10ul
Water Make up to 50ul

2. Use two-step (all ABI7700TaqMan quantitative analysis) or three-step PCR. For three-step PCR, run 40-50 cycles for amplification using the following conditions.



For two-step PCR, omit the 72°C extension and increase the annealing temperature to 60°C.
~The quantification depends on the HotStart program.

3. Continue with the optimization protocol in step 4 below.

III. Method B-SMARTCYCLER

1. Set up the following PCR components for each reaction in appropriate tubes. Mix the HotStart mixture with primers and probes/SYBRGreen, and add the template in the required amount. There are five 10-fold dilutions of template DNA.

2XHotStartMix 2.5ul
Primer A 0.3~0.8umol/L
Primer B 0.3~0.8umol/L
Probe or SYBRGreen 0.1~0.5umol/L
MgCl2(25 mmol/L) 3~6 mmol/L
Template DNA (500ng~50pg) 5ul
Water Make up to 25ul

The Smartcycler requires the use of specialized tubes.

2. Using two- or three-step PCR For three-step PCR, run 40 cycles for amplification using the following conditions.



For two-step PCR, omit the 72°C extension and increase the annealing temperature to 60°C. The annealing temperature should be increased to 60°C. The annealing temperature should be increased to 60°C.
~undefined depending on the HotStart program.

3. Continue with the optimization protocol in step 4 below.

IV.Methods C-----LIGHTCYCLERPCR

1. Set up the following PCR components for each reaction in appropriate tubes. Mix the HotStart mixture and primers and probes/SYBRGreen, adding the template in the required amounts. There are five 10-fold dilutions of template DNA.

2XFastStartMix 10ul
Primer A 0.1~0.5umol/L
Primer B 0.1~0.5umol/L
Probe or SYBRGreeundefined 0.1~0.5umol/L
MgCl₂ (25 mmol/L) 3~6 mmol/L
Template DNA(500ng~50pg) 5ul
Water Make up to 20ul

The ~undefinedLightCycler requires the use of a capillary tube.

2. Using three-step PCR, run 40 cycles of amplification under the following conditions.



3. Continue with the optimization protocol in step 4 below.
A practical approach to optimizing set-point analysis (see InnisandGelfand1990;HarrisandJones1997)

4. To optimize amplicons and primers, analyze the real-time PCR products using the unlinking curve function, which is not directly available on the ABI770. For individual products, the results should show that they have a specific unstranding peak with no secondary beacons indicating the formation of primer dimers. When the unstranding curve data is expressed as a first order derivative it will be a single peak. Multiple peaks indicate the presence of other amplification products. If multiple peaks are detected, it indicates that the magnesium ion concentration may need to be further optimized, but usually this indicates the need to redesign the primer.

5. To optimize the probe, a standard curve can be made using a 1-fold dilution of the target molecule in any unit of your choice. For example, assuming that the highest concentration of DNA is ¹⁰⁷, a series of 10-fold dilutions of DNA would be ¹⁰⁷, ¹⁰⁶, ¹⁰⁵, ¹⁰⁴, and ¹⁰³, respectively, and these values would be keyed in as a starting volume and used in the software to make a standard curve at the end of the quantitative analysis. Using the Cycling Threshold (Ct) value and the starting concentration of DNA it will be possible to calculate the reaction efficiency.

6. Similar to conventional PCR, further reactions with a series of 10-fold dilutions of standard solutions are required to optimize MgCl₂ concentration, primer concentration, probe concentration, annealing temperature, and to adjust the PCR protocol.

7. For systems that do not have their own rapid cycling protocols, shorter cycling protocols can be attempted to reduce the time for quantitative analysis.

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