title: LigAmp for sensitive detection of single-nucleotide differences.
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 approximately 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.
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
authors.forename: Chanjuan
authors.initials: C
authors.lastname: Shi
authors.forename: Susan H
authors.initials: SH
authors.lastname: Eshleman
authors.forename: Dana
authors.initials: D
authors.lastname: Jones
authors.forename: Noriyoshi
authors.initials: N
authors.lastname: Fukushima
authors.forename: Li
authors.initials: L
authors.lastname: Hua
authors.forename: Antony R
authors.initials: AR
authors.lastname: Parker
authors.forename: Charles J
authors.initials: CJ
authors.lastname: Yeo
authors.forename: Ralph H
authors.initials: RH
authors.lastname: Hruban
authors.forename: Michael G
authors.initials: MG
authors.lastname: Goggins
authors.forename: James R
authors.initials: JR
authors.lastname: Eshleman
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods*; D024882:Drug Resistance, Viral / Q000235:genetics; D005820:Genetic Testing / Q000379:methods*; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000378:metabolism; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment / Q000379:methods*; D017422:Sequence Analysis, DNA / Q000379:methods*; D018631:ras Proteins
chemical_list: D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins