4. Multiplex Polymerase Chain Reaction
Multiplex PCR is molecular biology technique for amplification of multiple targets in a single PCR reaction. Indeed, multiplex PCR is a modification of conventional or realtime
PCR in which two or more different PCR products are amplified within a single reaction (Henegariu, 1997). In a multiplexing assay, more than one target sequence can be amplified using multiple primer pairs in a reaction mixture. As an extension to practical use of PCR, this technique has potential to produce considerable time saving and effort within the laboratory without compromising on utility of the experiment
(Elnifro et. al., 2000). By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is considered …show more content…
After each cycle, the newly synthesized DNA strands can serve as template in the next cycle.
4.2. Advantages of Multiplex Polymerase Chain Reaction
Since its first description in 1988, multiplex PCR has been successfully applied in many areas of DNA testing including analyses of deletion, mutations, and polymorphisms, or quantitative assays and reverse transcription (Henegariu et al., 1997). Indeed, this technique has several advantages such as increasing the number of genes that can be analyzed, reducing sample requirement, saving time, effort, and saving potentially money. Furthermore, using multiplex PCR, the same reaction conditions can be provided for several targets. This method can also provide internal control within the same well and convenient for screening. In fact, in food safety and quality assurance, this method has become a promising alternative approach in detecting the sources of food. The presence of food borne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labeling in foods suitable for vegetarians are among the subjects where society demands total transparency. The use multiplex PCR in food industries is a way to satisfy …show more content…
Optimization of Multiplex Polymerase Chain Reaction
Multiplex PCR can be optimized by several ways. When optimizing a multiplex PCR assay, Taq DNA polymerase enzyme concentrations must be carefully considered.
Basically, too high concentration of Taq DNA polymerase enzyme concentration will produce non-specific background products, but if the concentration of enzyme is too low, an insufficient amount of desired product is produced. The cofactor MgCl2 concentration may also affect primer annealing, strand dissociation temperature of template and PCR product, product specificity, and enzyme activity (Innis and Gelfand,
1990). In fact, the specificity of PCR can be increased using lower dNTPs concentration. Low dNTPs concentration will minimize mis-priming at non-target sites and reduce the extending mis-incorporated nucleotides. Next, an applicable Ta is 5 oC below the true Tm of the primers. Increasing Ta enhances discrimination against incorrectly annealed primers and reduces misextension of incorrect nucleotides at the
3‟-end of primers (Innis and Gelfand, 1990; Rybicki, 2005). For these reasons, multiplex PCR are better performed using longer primers and high annealing and
extension
Multiplex PCR is molecular biology technique for amplification of multiple targets in a single PCR reaction. Indeed, multiplex PCR is a modification of conventional or realtime
PCR in which two or more different PCR products are amplified within a single reaction (Henegariu, 1997). In a multiplexing assay, more than one target sequence can be amplified using multiple primer pairs in a reaction mixture. As an extension to practical use of PCR, this technique has potential to produce considerable time saving and effort within the laboratory without compromising on utility of the experiment
(Elnifro et. al., 2000). By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is considered …show more content…
After each cycle, the newly synthesized DNA strands can serve as template in the next cycle.
4.2. Advantages of Multiplex Polymerase Chain Reaction
Since its first description in 1988, multiplex PCR has been successfully applied in many areas of DNA testing including analyses of deletion, mutations, and polymorphisms, or quantitative assays and reverse transcription (Henegariu et al., 1997). Indeed, this technique has several advantages such as increasing the number of genes that can be analyzed, reducing sample requirement, saving time, effort, and saving potentially money. Furthermore, using multiplex PCR, the same reaction conditions can be provided for several targets. This method can also provide internal control within the same well and convenient for screening. In fact, in food safety and quality assurance, this method has become a promising alternative approach in detecting the sources of food. The presence of food borne pathogens and their potential hazard, the use of genetically modified organisms (GMOs) in food production, and the correct labeling in foods suitable for vegetarians are among the subjects where society demands total transparency. The use multiplex PCR in food industries is a way to satisfy …show more content…
Optimization of Multiplex Polymerase Chain Reaction
Multiplex PCR can be optimized by several ways. When optimizing a multiplex PCR assay, Taq DNA polymerase enzyme concentrations must be carefully considered.
Basically, too high concentration of Taq DNA polymerase enzyme concentration will produce non-specific background products, but if the concentration of enzyme is too low, an insufficient amount of desired product is produced. The cofactor MgCl2 concentration may also affect primer annealing, strand dissociation temperature of template and PCR product, product specificity, and enzyme activity (Innis and Gelfand,
1990). In fact, the specificity of PCR can be increased using lower dNTPs concentration. Low dNTPs concentration will minimize mis-priming at non-target sites and reduce the extending mis-incorporated nucleotides. Next, an applicable Ta is 5 oC below the true Tm of the primers. Increasing Ta enhances discrimination against incorrectly annealed primers and reduces misextension of incorrect nucleotides at the
3‟-end of primers (Innis and Gelfand, 1990; Rybicki, 2005). For these reasons, multiplex PCR are better performed using longer primers and high annealing and
extension