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LigAmp for sensitive detection of single-nucleotide differences

Nature Methods volume 1, pages 141–147 (2004)Cite this article

Abstract

We developed the LigAmp assay for sensitive detection and accurate quantification of viruses and cells with single-base mutations. In LigAmp, two oligonucleotides are hybridized adjacently to a DNA template. One oligonucleotide matches the target sequence and contains a probe sequence. If the target sequence is present, the oligonucleotides are ligated together and detected using real-time PCR. LigAmp detected KRAS2 mutant DNA at 0.01% in mixtures of different cell lines. KRAS2 mutations were also detected in pancreatic duct juice from patients with pancreatic cancer. LigAmp detected the K103N HIV-1 drug resistance mutation at 0.01% in plasmid mixtures and at ∼0.1% in DNA amplified from plasma HIV-1. Detection in both systems is linear over a broad dynamic range. Preliminary evidence indicates that reactions can be multiplexed. This assay may find applications in the diagnosis of genetic disorders and the management of patients with cancer and infectious diseases.

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Figure 1: Overview of the LigAmp assay.
Figure 2: Detection of genomic DNA containing the KRAS2 mutation.
Figure 3: Detection of mutant KRAS2 sequences in pancreatic duct juice from pancreatic cancer patients.
Figure 4: Detection of the HIV-1 K103N mutation in plasmid mixtures.
Figure 5: Population sequencing and LigAmp analysis of HIV-1 in plasma samples.

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Acknowledgements

We thank A. Maitra, B. Wendelburg (Cepheid), J.B. Jackson, K. Kinzler, B. Vogelstein, K. Murphy, M. Slater, J. Strain (Applied Biosystems) and K. Brune for helpful discussions; J. Mellors (University of Pittsburgh) for providing plasmids containing HIV-1 molecular clones with and without the K103N mutation; and S. Hudelson for excellent technical assistance. This work was supported by grant R01 CA81439 from the National Cancer Institute, and the Maryland Cigarette Restitution Fund (to J.R.E.), by the HIV Prevention Trials Network sponsored by the National Institute for Allergies and Infectious Diseases (NIAID), National Institutes of Child Health and Human Development, National Institute on Drug Abuse, National Institute of Mental Health and Office of AIDS Research of the National Institutes of Health (NIH), Department of Health and Human Services (U01-AI-46745 and U01-AI-48054) and the Adult AIDS Clinical Trials Groups (NIH, Division of AIDS, NIAID, U01-AI-38858) and R01-HD042965-01 (to S.H.E.), by National Cancer Institute SPORE grant P50-CA-62924, and by a Fellowship from the Canadian Institute of Health Research (to C.S.).

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Authors and Affiliations

  1. Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, 21205, Maryland, USA

    Chanjuan Shi, Susan H Eshleman, Dana Jones, Noriyoshi Fukushima, Li Hua, Antony R Parker, Ralph H Hruban, Michael G Goggins & James R Eshleman

  2. Department of Surgery, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, 21205, Maryland, USA

    Charles J Yeo & Michael G Goggins

  3. Department of Medicine, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, 21205, Maryland, USA

    Michael G Goggins

  4. Department of Oncology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, 21205, Maryland, USA

    Ralph H Hruban & James R Eshleman

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  1. Chanjuan Shi
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Correspondence to James R Eshleman.

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Supplementary information

Supplementary Table 1

Oligonucleotides and probes (PDF 21 kb)

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Shi, C., Eshleman, S., Jones, D. et al. LigAmp for sensitive detection of single-nucleotide differences. Nat Methods 1, 141–147 (2004). https://doi.org/10.1038/nmeth713

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