The source of SYBR green master mix determines outcome of nucleic acid amplification reactions.

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

doi:10.1186/s13104-016-2093-4

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

Highlights

Quantitative (q) polymerase chain reaction (PCR) by amplification of nucleic acid with a fluorescent dye is widely used
Sieber and colleagues have shown substantial performance discrepancies among commercial complementary DNA (cDNA) synthesis kits and quantitative polymerase chain reaction (qPCR) kits in three species [20]; the current study mainly focused on the RT-qPCR process, thereby including specificity of the PCR assays as an essential outcome parameter
Our data show that three commercial PCR mixes exhibit significant differences with respect to sensitivity of the PCR assay when applying a large panel of primer sets for mRNA transcript quantitation

Summary

Introduction

Quantitative (q) PCR by amplification of nucleic acid with a fluorescent dye is widely used. Real-time polymerase chain reaction (PCR) is widely used to measure gene expression and DNA copies [1, 2]. The most commonly used methods for quantitative polymerase chain reaction (qPCR) are based on non-specific SYBR green chemistry and specific Taqman probe chemistry [3]. The combination of a saturating, DNA-binding dye with superior instrumentation and sophisticated software enables the detection of genetic variations by analyzing PCR melting curves at a finer temperature resolution [11]. HRM reactions generate specific and sensitive melting profiles. They can be used for genotyping, mutation screening, and methylation analysis based on heterozygosity, length, and GC content [13]

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