The source of SYBR green master mix determines outcome of nucleic acid amplification reactionsReport as inadecuate




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BMC Research Notes

, 9:292

Molecular Biology

Abstract

BackgroundQuantitative q PCR by amplification of nucleic acid with a fluorescent dye is widely used. Selection of adequate PCR reagents and devices is relevant to achieve reliable and consistent data. Our main objective was to test the robustness of different commercial SYBR green PCR mixes with respect to specificity and sensitivity of the PCR assay, across various PCR machines Light Cycler 96, ViiA7 and amplification protocols. Herein, we applied PCR protocols for determining mRNA transcript levels, DNA copy numbers, and DNA genotype.

ResultsFirst, we set up 70 primer-based assays that targeted immune-related mRNA transcripts. Of the 70 assays 66 94.3 % resulted in a single melting curve peak, indicating specificity of the amplification, with PCR mixes from large vendors Roche, ABI, Bio-Rad. But this was only seen when the PCR protocol that was indicated in the vendor’s guidelines for each particular mix was applied. When deviating from the prescribed protocol, suboptimal melting curves were most often seen when using Roche SYBR green. With respect to PCR yields, the use of ABI mix more often led to lower Cq values. Second, we set up 20 primer-selective PCR assays to target different insertion-deletion and single nucleotide polymorphism regions throughout the genome. The variation in delta Cq between positive and negative DNA samples among the PCR assays was the lowest when using ABI master mix. Finally, the quality of high resolution melting HRM assays for DNA genotyping was compared between four commercial HRM PCR mixes Roche, Bioline, PCR Biosystems, ABI. Only Roche and ABI mixes produced optimal clusters of melting profiles that clearly distinguished genotype variants.

ConclusionsThe current results show a preference for the use of ABI mix when it comes to obtaining higher sensitivity in cDNA analysis and a higher consistency among assays in distinguishing DNA genotypes among different individuals. For HRM assays, it is advisable to use master mix from a relatively large vendor.

KeywordsQuantitative PCR Specificity Genomic DNA mRNA High resolution melting Performance AbbreviationsHRMhigh resolution melting

SNPsingle nucleotide polymorphism

PCRpolymerase chain reaction

qPCRquantitative PCR

DNAdeoxyribonucleic acid

Ds DNAdouble-stranded DNA

cDNAcomplementary DNA

gDNAgenomic DNA

ABIapplied biosystems

LC 96light cycler 96

NTCno template control

−RT controlminus reverse transcriptase control

Electronic supplementary materialThe online version of this article doi:10.1186-s13104-016-2093-4 contains supplementary material, which is available to authorized users.

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Author: Jianxin Yang - Berit Kemps-Mols - Marijke Spruyt-Gerritse - Jacqueline Anholts - Frans Claas - Michael Eikmans

Source: https://link.springer.com/







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