LigAmp for sensitive detection of single-nucleotide differences.
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.
Shi
Chanjuan
C
Department of Pathology, Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, Maryland 21205, USA.
Eshleman
Susan H
SH
Jones
Dana
D
Fukushima
Noriyoshi
N
Hua
Li
L
Parker
Antony R
AR
Yeo
Charles J
CJ
Hruban
Ralph H
RH
Goggins
Michael G
MG
Eshleman
James R
JR
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
D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins