LigAmp for sensitive detection of single-nucleotide differences

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

Research output: Contribution to journalArticlepeer-review

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.

Original languageEnglish (US)
Pages (from-to)141-147
Number of pages7
JournalNature Methods
Volume1
Issue number2
DOIs
StatePublished - Nov 2004

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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