Abstract
In this study, a novel universal primer-multiplex-PCR (UP-M-PCR) method adding a universal primer (UP) in the multiplex PCR reaction system was described. A universal adapter was designed in the 5′-end of each specific primer pairs which matched with the specific DNA sequences for each template and also used as the universal primer (UP). PCR products were analyzed on sequencing gel electrophoresis (SGE) which had the advantage of exhibiting extraordinary resolution. This method overcame the disadvantages rooted deeply in conventional multiplex PCR such as complex manipulation, lower sensitivity, self-inhibition and amplification disparity resulting from different primers, and it got a high specificity and had a low detection limit of 0.1 ng for single kind of crops when screening the presence of genetically modified (GM) crops in mixture samples. The novel developed multiplex PCR assay with sequencing gel electrophoresis analysis will be useful in many fields, such as verifying the GM status of a sample irrespective of the crop and GM trait and so on.
Highlights
Nucleic acid analysis has become increasingly important in a variety of applications, such as the genotyping of individuals, the detection of infectious diseases, tissue typing for histocompatability, identifying individuals in forensic diagnosis, paternity testing, and monitoring the genetic make-up of plants and animals in agricultural breeding programs [1]
The fifteen-target universal primer (UP)-M-polymerase chain reaction (PCR) we developed contains the most targets in all the multiplex PCR methods reported before, and covers most selectable marker and reporter genes commonly employed in genetically modified (GM) crops, which will totally meet the demand of verifying the GM status of a sample irrespective of the crop and GM trait
Feasibility of Universal Primer (UP) Keeping the concentration of templates at 50 ng, with the amount of the specific primers for Lec gene decreasing (500 nmol L21, 50 nmol L21, 25 nmol L21, 5 nmol L21 ), the intensity of band fell down markedly (25 nmol L21) until to nothing (5 nmol L21) in conventional singlet PCR (Figure 2, lanes 1, 2, 3, 4), which showed that the concentration of amplified fragments became lower and lower
Summary
Nucleic acid analysis has become increasingly important in a variety of applications, such as the genotyping of individuals, the detection of infectious diseases, tissue typing for histocompatability, identifying individuals in forensic diagnosis, paternity testing, and monitoring the genetic make-up of plants and animals in agricultural breeding programs [1]. Techniques based on polymerase chain reaction (PCR) provide a powerful tool for the amplification of minute amounts of initial target sequences. Most PCR protocols involve reactions that amplify a single target. Multiplex PCR is a variation of the conventional technique in which two or more targets are simultaneously amplified in the same reaction. This approach has the potential for greater reliability, flexibility, and cost reduction. As far as we know, nine-target multiplex PCR method has been reported to simultaneously detect eight maize lines as well as the endogenous Zein gene in a single reaction tube [2], which contains the most targets in reported multiplex-PCR methods
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