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Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR)

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Abstract

Background

Polymerase chain reaction (PCR) is widely used in biological research and diagnostics because of its high sensitivity and specificity. However, the sensitivity of PCR is strongly influenced by topological characteristics of the template. Supercoiled templates are known to inhibit PCR, whereas linearized forms of the same supercoiled templates facilitate PCR.

Objectives

This study was conducted to compare the PCR efficiency of circular supercoiled DNA templates to their restriction endonuclease (RE)-mediated linearized forms. Additionally, we also evaluated the possibility of RE digestion of the circular supercoiled templates within the complete PCR buffer.

Methods

Following a systematic approach, we demonstrated that circular supercoiled templates could be efficiently linearized by RE in the complete PCR buffer itself. This allowed linearization of circular supercoiled templates and their subsequent amplification in the PCR buffer in a single-tube format.

Results

Using this extremely simple RE-PCR approach, we documented up to tenfold increases in detection efficiency of PCR with two different circular supercoiled templates of clinical origin, including an international calibration standard.

Conclusions

This inexpensive and easy approach to increasing PCR sensitivity can be easily adapted to any standard PCR protocol aimed at amplifying circular supercoiled genomes. Apart from its application in the development of sensitive clinical diagnostic PCR assays for a large number of organisms, this method could also prove to be very useful in simplifying the existing protocols for other applications where pre-PCR restriction digestion is required, such as mutation detection, genotyping, and selective template amplification.

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Acknowledgments

The authors graciously thank Dr. Prerna Guleria, for her help at different points of this work.

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Authors and Affiliations

  1. Molecular Virology Laboratory, Defence Research Laboratory (DRDO), Tezpur, Assam, 784001, India

    Sibnarayan Datta, Raghvendra Budhauliya, Soumya Chatterjee,  Vanlalhmuaka & Vijay Veer

  2. Hepatitis Research Laboratory, ICMR Virus Unit, Kolkata (ICMR), Kolkata, 700010, West Bengal, India

    Runu Chakravarty

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  1. Sibnarayan Datta
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Correspondence to Sibnarayan Datta.

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SD, RB, SC, VM, VV, and RC have no conflicts of interest that are directly relevant to the content of this manuscript.

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This work was supported by funds from the Defence Research and Development Organization (DRDO), Indian Council of Medical Research (ICMR), and the University Grants Commission (UGC), Government of India.

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Datta, S., Budhauliya, R., Chatterjee, S. et al. Enhancement of PCR Detection Limit by Single-Tube Restriction Endonuclease-PCR (RE-PCR). Mol Diagn Ther 20, 297–305 (2016). https://doi.org/10.1007/s40291-016-0195-2

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