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Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing

Nature Protocols volume 2, pages 2429–2438 (2007)Cite this article

Abstract

This protocol describes the design and execution of monoplex and multiplex linear-after-the-exponential (LATE)-PCR assays using a novel reagent, PrimeSafe, that suppresses all forms of mispriming. LATE-PCR is an advanced form of asymmetric amplification that uses a limiting primer and an excess primer for efficient exponential amplification of double-stranded DNA, followed by linear amplification of one strand. Each single-stranded amplicon can be quantitatively detected in real time or at end point. By separating primer annealing from product detection, LATE-PCR enables product analysis at low temperatures. Alternatively, each single strand can be sequenced by a convenient Dilute-'N'-Go procedure. Amplified samples are diluted with individual sequencing primers without the use of columns or spins. We have amplified and then sequenced 15 different single-stranded products generated in a single multiplexed LATE-PCR comprised of 15 pairs of unrelated primers. Dilute-'N'-Go dideoxy sequencing is more convenient, faster and less expensive than sequencing double-stranded amplicons generated via conventional symmetric PCR. The preparation of LATE-PCR products for Dilute-'N'-Go sequencing takes only 30 seconds.

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Figure 1: Use of SYBR Green dye and a low-Tm Cy5-labeled ResonSense probe permits detection of both double-stranded and single-stranded DNA products in LATE-PCR assays.
Figure 2: Detection of LATE-PCR amplification products in samples containing different initial concentrations of DNA using a low-Tm molecular beacon, labeled with FAM (6-carboxylfluorescein).
Figure 3: Effect of adding 0.4× PrimeSafe-045 to a symmetric PCR on product specificity (determined by agarose gel electrophoresis) and the SYBR Green kinetic plot under conditions that promote substantial mispriming (use of native Taq polymerase and incubation at room temperature for 30 min before the start of PCR).
Figure 4: Example of a LATE-PCR pentaplex amplification.
Figure 5: In silico melt curves from Visual OMP (DNA software) for 15 pairs of primers used for a 15-plex LATE-PCR assay.
Figure 6: An example of how to determine the number of linear LATE-PCR cycles required to generate sufficient single-stranded DNA for Dilute-'N'-Go dideoxy sequencing.
Figure 7: Example of LATE-PCR Dilute-'N'-Go cycle sequencing.
Figure 8: Dilute-'N'-Go dideoxy sequencing of multiplexed amplification products shown in Figure 4.

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Authors and Affiliations

  1. Department of Biology, Brandeis University, Waltham, 02454-9110, Massachusetts, USA

    John E Rice, J Aquiles Sanchez, Kenneth E Pierce, Arthur H Reis Jr, Adam Osborne & Lawrence J Wangh

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  1. John E Rice
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  2. J Aquiles Sanchez
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  3. Kenneth E Pierce
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  6. Lawrence J Wangh
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Correspondence to Lawrence J Wangh.

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Competing interests

PrimeSafe® is an independent discovery of the Wangh Laboratory that has been licensed to Smiths Detection by Brandeis University. The inventors of this technology will receive a fraction of any future royalties received by Brandeis University through the sale of PrimeSafe®. The research described here has been supported in part by a grant from Smiths Detection.

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Rice, J., Sanchez, J., Pierce, K. et al. Monoplex/multiplex linear-after-the-exponential-PCR assays combined with PrimeSafe and Dilute-'N'-Go sequencing. Nat Protoc 2, 2429–2438 (2007). https://doi.org/10.1038/nprot.2007.362

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