Real-time PCR (qPCR) and allele-specific probes are important technologies in gene quantification and detection of specific genetic mutations. These technologies have high accuracy and specificity in gene analysis and diagnosis and are used in many research and medical fields.
1. Real-time PCR (qPCR)
Real-time PCR (quantitative PCR, qPCR) is a technology that amplifies specific sequences of DNA or RNA and quantitatively measures the amplification process in real time. Unlike conventional PCR, qPCR allows the amount of amplified product to be confirmed in real time and the amount of target DNA or RNA to be accurately measured.
- Basic Principle
In qPCR, a DNA sample is amplified in cycles and the amount of amplified product is detected using a fluorescent dye or probe. qPCR monitors the progress of amplification by measuring the amount of fluorescence per cycle in real time, since the fluorescence intensity increases in proportion to the amount of amplified DNA. - Applications
- Pathogen detection: Used in the diagnosis of infectious diseases, the fluorescent probe can quickly and accurately detect the presence of pathogens such as viruses and bacteria.
- Gene expression analysis: Evaluates the extent to which a particular gene is expressed by measuring the amount of mRNA and quantifying the level of gene expression.
- Detection of genetic mutations: It is also used to detect genetic mutations associated with cancer and genetic diseases.
Types of fluorescent probes
In qPCR, fluorescent probes are used to detect amplified products. Typical probes are SYBR Green and TaqMan probes, which bind to DNA double-strands and emit fluorescence, and TaqMan probes, which bind specifically to a particular sequence and emit fluorescence.
2. Allele-specific probes
Allele-specific probes are probes designed to detect specific gene mutations or polymorphisms (SNPs) and have high specificity for the allele of interest (different variants of a gene). Allele-specific probes can be used to distinguish samples with different alleles and precisely detect the presence or absence and type of genetic variants.
- Basic Principle
Allele-specific probes are designed to bind only to the sequence of the specific allele of interest. A fluorescent dye is incorporated into the probe and a fluorescent signal is detected when the probe binds to the target sequence. This allows one to determine if a specific allele is present. - Applications
- Detection of genetic mutations: Used to detect specific alleles associated with cancer and genetic diseases.
- Evaluation of drug response: Used to identify polymorphisms in genes involved in drug metabolism and to select the appropriate treatment for an individual.
- SNP typing: used to analyze genetic polymorphisms (SNPs) and to investigate an individual’s genetic background.
Combination of qPCR and allele-specific probes
By combining qPCR with allele-specific probes, specific gene mutations and polymorphisms can be quantitatively measured with high precision. For example, using allele-specific probes for mutant and wild-type (normal allele) DNA amplified by qPCR allows quantitative confirmation of the presence or absence and percentage of mutations, which is important in the diagnosis of cancer and genetic diseases.
Conclusion
Real-time PCR (qPCR) is a technology that quantitatively analyzes DNA and RNA in real time while amplifying them, and is widely used for diagnosis of infectious diseases, gene expression analysis, and detection of genetic mutations. Allele-specific probes, on the other hand, are used to identify specific genetic mutations and polymorphisms with high precision, and when combined with qPCR, genetic information can be analyzed more accurately. This allows them to play an important role in disease diagnosis, personalized medicine, and research.
