Real-time fluorescence quantitative PCR-FP
Real-time fluorescence quantitative PCR-FP
Real-time fluorescence quantitative PCR-FP
Principle
Fluorescently labeled oligonucleotide probes are small molecules with weakly polarized light. When the probe binds to a specific fragment on the DNA, the molecular weight increases and the polarized light is enhanced. During PCR amplification, as the primer-mediated formation of a new strand occurs, the probe dissociates from the template and becomes free again, and the polarized light decreases again.
During the annealing phase of each PCR cycle, the probe can be repaired to the DNA strand and the polarized light is enhanced. Detecting the increase in polarized light at this stage allows for quantitative detection. When the amplified nucleic acid sequence does not contain specific DNA fragments that are compatible with the probe, the fluorescent probe cannot be paired with the nucleic acid sequence, and its fluorescence polarization value is very weak. Therefore, it is possible to detect whether the amplified nucleotide sequence contains the target DNA fragments.
Operation method
Real-time fluorescence quantitative PCR-FP technology
Principle
Fluorescently labeled oligonucleotide probes are small molecules with weakly polarized light. When the probe binds to a specific fragment on the DNA, the molecular weight increases and the polarized light is enhanced. During PCR amplification, as the primer-mediated formation of a new strand occurs, the probe dissociates from the template and becomes free again, and the polarized light decreases again. During the annealing phase of each PCR cycle, the probe can be repaired to the DNA strand and the polarized light is enhanced. Detecting the increase in polarized light at this stage allows for quantitative detection. When the amplified nucleic acid sequence does not contain specific DNA fragments that are compatible with the probe, the fluorescent probe cannot be paired with the nucleic acid sequence, and its fluorescence polarization value is very weak. Therefore, it is possible to detect whether the amplified nucleotide sequence contains the target DNA fragments.
Materials and Instruments
MJ Research V2.0; Victor II fluorescence polarization detector; dNTPs, Taq DNA polymerase, serum specimens, primers and probes. Move 1. Serum treatment: Add 30 μl of treatment solution (10 mmol/L Tris-HCl, pH 8.0 1 mmol/L EDTA, 1 ml/L NP40) to 30 μl of serum specimen and boil for 15 min in a centrifuge tube, centrifuge for 5 min at 7,000 r/min, and then aspirate 2 μl of supernatant to be used as templates. 2, PCR amplification reaction and liquid hybridization: take 2 μl of template and add it to 28 μl of PCR reaction solution (10×PCR buffer 3 μl, dNTPs 160 μmol/L, upstream universal primer C1 and downstream universal primer C2 3 pmol each, probe 1.5 pmol, Taq DNA polymerase 1 U). PCR program: 94 ℃, 2 min and then enter into the loop PCR program: 94 ℃, 2 min, then enter the cycle, 94 ℃, 5 s, 60 ℃, 55 s, 72 ℃, 45 s, cycle 25 times, and finally 94 ℃, 5 s, 45 ℃, 20 s. The last cycle was 94 ℃, 5 s, 45 ℃, 20 s. 3. Probe hybridization: 10 μl of the hybridized PCR product was added to 45 μl of hybridization buffer containing 1 g/L polyethylene glycol 4,000, 300 ml/L DMSO, 6× SSC (pH 10), 0.01 mol/L sodium phosphate (pH 8.0), 1 mmol/L EDTA (pH 8.0), 5 g/L SDS, 100 μg/ml denatured The fluorescence polarization was detected by using Victor Ⅱ Fluorescence Polarization Detector to detect the polarization value of fluorescein R110. 4. Calculation of determination criteria: In this experimental protocol, for example, 10 HBV DNA negative sera and 10 HBV DNA positive sera were randomly selected for testing. cut off value = average polarized light value of the negative sample results + 10% × average polarized light value of the positive sample results. Samples with polarization values > 110% of the cut off value and < 90% of the cut off value were considered positive, while samples within 2 thresholds (including the threshold value) were considered +/-. The judgment criteria are: cut off = 60 mp, then the negative and positive criteria are 54 and 66 mp. Caveat 1. In FP detection, there is a limit to the size of the labeled molecule, and increasing the molecular weight for a given fluorophore will reduce the potential detection window;2, The allowable size of the labeled molecule also depends on the fluorescence lifetime of the label;3, Fluorescent labeled molecule size ＜1.5 kD is optimal. For more product details, please visit Aladdin Scientific website.