Multiplex PCR
Multiplex PCR is a technique that uses a polymerase chain reaction to amplify several different DNA sequences simultaneously, as if performing many single PCR reactions in one reaction system. Multiplex PCR was first used in 1988 to detect deletions in the dystrophin gene and later for the analysis of steroid sulfatase gene deletions. Multiplex PCR has many advantages, such as saving reagents, time, and samples, increasing detection efficiency and sensitivity, obtaining more information, and providing internal controls. Multiplex PCR also faces some challenges, such as primer design, reaction optimization, amplification bias, non-specific amplification, etc. Multiplex PCR has a wide range of applications in different fields, such as pathogen identification, SNP genotyping, mutation analysis, genetic disease diagnosis, template quantitation, linkage analysis, RNA detection, forensic science, and diet analysis.
The Basic Principle and Key Steps of Multiplex PCR
The basic principle of multiplex PCR is to use a polymerase chain reaction to amplify several different DNA sequences simultaneously, each corresponding to a pair of specific primers. This technique requires two or more probes that can be distinguished from each other and detected simultaneously. There are various probe technologies, all using fluorescent dyes. Multiplex PCR can be divided into two types: single-template multiplex PCR and multi-template multiplex PCR. The principle of single-template multiplex PCR is to use one template (such as genomic DNA or cDNA) and multiple pairs of primers in one reaction system, amplifying fragments of different lengths or locations. This type of multiplex PCR is often used for genetic disease diagnosis, gene deletion analysis, linkage analysis, etc. The principle of multi-template multiplex PCR is to use different types of templates (such as different pathogens or different species of DNA) and multiple pairs of primers in one reaction system, amplifying specific fragments. This type of multiplex PCR is often used for pathogen identification, SNP genotyping, food analysis, etc.
The key steps of multiplex PCR are primer design, reaction system and amplification condition optimization, amplification product detection, and analysis. Primer design requires that the primers have no complementarity or secondary structure between them, high specificity and appropriate annealing temperature with the template, similar primer length and GC content, and appropriate primer concentration. The reaction system requires the use of polymerase, buffer, dNTPs, MgCl2, etc. suitable for multiplex PCR and adjustments according to different templates and primers. Amplification conditions require the selection of an appropriate cycle number, annealing temperature, extension time, etc., to avoid non-specific amplification or amplification bias. Amplification product detection and analysis require the use of different colors of fluorescent probes or electrophoresis gel to distinguish fragments of different lengths or locations and perform quantitative or qualitative evaluation.
The Main Characteristics of Multiplex PCR
Multiplex PCR is a technique that allows the simultaneous amplification and detection of multiple DNA sequences in a single reaction. It has several advantages over single PCR, such as higher efficiency, sensitivity, specificity, and diversity. Multiplex PCR can provide more information with less sample and time, as well as internal controls for quality assurance. However, multiplex PCR also has some challenges, such as the need for careful primer design and reaction optimization, the risk of amplification bias and non-specific amplification, and the difficulty of modification or adjustment. Multiplex PCR has a wide range of applications in different fields, such as genetic disease diagnosis, pathogen identification, SNP genotyping, and food analysis.
Table 1. Comparison of Multiplex PCR and Single PCR
| Multiplex PCR | Single PCR |
|---|---|
| Amplify multiple target sequences simultaneously | Amplify only one target sequence |
| Require multiple pairs of specific primers and different colors of fluorescent probes | Require only one pair of primers and one fluorescent probe |
| Save reagents, time, and samples; Increases detection efficiency and sensitivity; Obtain more information; and provide internal controls | Consume more reagents, time, and samples; decrease detection efficiency and sensitivity; obtain only single information; and lack internal controls |
| Have higher difficulty in primer design, reaction optimization, amplification bias, non-specific amplification, etc., and require multiple rounds of validation | Have lower difficulty in primer design, reaction optimization, amplification bias, non-specific amplification, etc., and require less validation |
| Hard to modify or adjust | Easy to modify or adjust |
Applications of Multiplex PCR
Multiplex PCR has a wide range of applications in different fields, mainly including the following aspects: genetic disease diagnosis, pathogen identification, SNP genotyping, and food analysis. Multiplex PCR can simultaneously detect multiple genes or mutations related to genetic diseases, such as Duchenne muscular dystrophy, steroid sulfatase deficiency, galactosemia, etc. Multiplex PCR can improve the efficiency and accuracy of genetic disease diagnosis, save samples and costs, and provide internal controls. Multiplex PCR can also simultaneously detect specific markers of various pathogens, such as bacteria, fungi, parasites, viruses, etc. Multiplex PCR can quickly, sensitively, and specifically identify pathogens, which is helpful for clinical diagnosis and treatment. Multiplex PCR can also simultaneously amplify multiple SNP loci, perform gene typing, and perform genetic diversity analysis. Multiplex PCR can increase the throughput and reliability of SNP genotyping and is suitable for large-scale population genetics and association studies. Multiplex PCR can also simultaneously detect various ingredients or contaminants in food, such as genetically modified components, animal species, pathogenic bacteria, etc. Multiplex PCR can effectively monitor the quality and safety of food and protect the health and interests of consumers. In addition to these aspects, multiplex PCR has other applications, such as forensic science, template quantitation, linkage analysis, RNA detection, etc. Multiplex PCR is a powerful and flexible technique that can be designed and optimized according to different purposes and needs.
References
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