PCR-CE stands for Polymerase Chain Reaction - Capillary Electrophoresis. It is a hybrid molecular biology technique that combines the high-efficiency amplification capabilities of PCR with the high-resolution separation and detection capabilities of CE. PCR is used to specifically amplify target DNA fragments. The amplified products are then separated and detected by capillary electrophoresis, enabling qualitative, quantitative, or length analysis of the amplicons.
Working Principle
Sample Preparation and PCR Amplification: DNA is extracted from the sample, and PCR amplification is performed using primers designed for the specific target. Fluorescent labels are often introduced directly during the amplification process (e.g., using fluorescently labeled primers).
Capillary Electrophoresis Injection: PCR products are injected into one end of a capillary tube.
Electrophoretic Separation: Under a high-voltage electric field, DNA fragments migrate through a sieving polymer within the capillary tube, physically separating them based on molecular weight.
Fluorescence Detection and Data Analysis: As DNA fragments of varying sizes pass through the detection window at the end of the capillary tube, laser light excites the attached fluorescent dye, generating a signal. Software records the signal and generates an electrophoresis profile, with the horizontal axis representing time (which can be converted to fragment size) and the vertical axis representing fluorescence intensity. By analyzing the position and height (or area) of the peaks, the size and relative abundance of the target fragments can be determined.
Main Applications of PCR-CE
MSI or STR Analysis：
Forensic Identification: DNA fingerprinting is the gold standard for paternity testing and criminal database development.
Tumor Diagnosis: Detection of Microsatellite Instability (MSI) is used for Lynch syndrome screening and immunotherapy efficacy prediction.
Genotyping:
Single Nucleotide Polymorphism (SNP) Analysis: Primers are designed to amplify fragments containing the SNP site, and their length specificity can be used for genotyping.
Indel Polymorphism Analysis: Genotyping is performed directly based on differences in PCR product length.
Mutation Detection: Used to detect known insertion/deletion mutations.
Gene Expression Analysis (Relative Quantification): cDNA is amplified by reverse transcription-PCR (RT-PCR), separated by CE, and relative quantification is performed by comparing the peak height or area of the amplified product of a specific gene in different samples.
Viral Load Analysis: Relative quantification of viral DNA is performed by competitive PCR or internal control calibration.
Advantages
High Resolution
Can distinguish DNA fragments that differ by only 1-4 bp in length, with far greater precision than agarose gel electrophoresis.
High-Throughput Automation
Automated sample loading in 96-well or 384-well plates allows for unattended, continuous analysis of large numbers of samples, resulting in extremely high efficiency.
Results are output as electropherograms, providing precise data, ease of software analysis, and archiving, with high objectivity.
No gel preparation required
This avoids the risk of exposure to carcinogens such as ethidium bromide (EB) during agarose gel electrophoresis.
Limitations
Relative Quantification
Typically only relative quantification (e.g., comparison between samples) is possible, not absolute quantification like qPCR..
High Cost
The cost of instrumentation and consumables (capillaries, sieving gel) is higher than that of conventional gel electrophoresis.
Lack of Real-Time Monitoring
Unlike qPCR, PCR-CE is an endpoint assay and can only be analyzed after the PCR is complete, preventing real-time observation of the amplification process.
Fragment size limitation
Optimal separation typically occurs within the tens to hundreds of base pairs, and separation efficiency may be reduced for very large fragments (>1000 base pairs).
Limited information
It primarily provides fragment length information and cannot directly detect specific changes in the sequence itself (such as point mutations that do not alter length), requiring integration with other techniques (such as sequencing).
Conclusion
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In summary, PCR-CE is a powerful endpoint analysis technology. Its core value lies in high-resolution, high-throughput fragment size analysis of PCR amplification products.
It is like first using PCR, like "precision scissors," to snip specific fragments from the vast DNA blueprint.
Then, using CE, like a "super-precise sieve," the lengths of these fragments are precisely measured.
Although it is inferior to qPCR in absolute quantification and real-time monitoring, and inferior to sequencing in interpreting detailed sequence information, it remains an indispensable core technology platform for applications based on fragment length differences (such as STR, MSI, and InDel typing) due to its automation, high precision, and high throughput.
