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The Construction And Application Of The PCR Primer Specificity Evaluating System And Multiplex PCR Primer Design System

Posted on:2010-08-15Degree:MasterType:Thesis
Country:ChinaCandidate:Z Y ShenFull Text:PDF
GTID:2120360275462377Subject:Biochemistry and Molecular Biology
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Polymerase Chain Reaction(PCR) is widely used in biological researches.The non-specific DNA products in PCR experiment are still a problem and seriously affect the efficiency of routine PCR and especially multiplex PCR because non-specific binding of the primers to heterogeneous DNA templates in a single tube. However,the problem of designing efficient multiplex PCR primers had been demonstrated to be a NP-complete problem by transformation to the 'Multiple Choice Matching' problem and can only obtain an efficient approximative algorithm. For example,a genetic algorithm can efficiently solve the multi-objective optimization problems for multiplex PCR primer design.In fact,the PCR primer design for multiple sequences of DNA templates based on two ways.The first step is to design a primer pair for each DNA template sequence and the second step is to design degenerate primers for multiple sequences.Although there are many factors could affect the results of multiplex PCR,one of the key points is to improve the specificity of the primer.Until now there is still a lack of efficient computer tools for primer pair's specificity analysis because of the complicated conditions of multiplex PCR reaction.Our work mainly focused on the nonspecific amplicons and developed a system called Primer Specificity Checking(PSC) for PCR primer specificity checking based on the NCBI Blast program and the genomic and cDNA databases of widely used species.and also the optimal parameter of word size is suggested for evaluation after testing various of primer pairs based on different species.Focusing on prediction of the possible non-specific amplicons,PSC can be used to optimize primer design or analyze the unexpected PCR results to improve the PCR efficiency.After then, combined with the feedback opinions of PSC,it was updated to MFEprimer for evaluating the specificity of PCR primers based on multiple factors including sequencing similarity,stability at the 3'- end of the primer,melting temperature,ion concentration etc.All these changes made it scientific and rational.After the paper published,MFEprimer as an evaluation tool for primer specificity had been accepted by international recognition.After do these,we further developed a multiplex PCR primer design program named MPprimer,which is mainly a combination of the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer.MPprimer will help the user to reduce the unspecific amplicons in the multiplex PCR experiments,so as to improve the reliability of multiplex PCR.In addition,we introduced a formula to calculate the mobility of all of the amplicons to produce a virtual electrophoretogram for the multiplex PCR reaction to allow the user have a glance before running the real PCR reaction.Five mouse genes(beta- actin,B2m,Pgk1,GAPDH,Rpll3a) and four C.elegan genes (F21A3.6,C26C6.7,act-1,R13A1.8) have been used to design primers by MPprimer for multiplex PCR respectively.The experimental evidences showed that MPprimer is a valuable tool for the user to improve the efficiency of multiplex PCR analysis.
Keywords/Search Tags:Primer3, MFEprimer, MPprimer, primer pair specificity combination, multiplex PCR
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