title: One-Step Ligation on RNA Amplification for the Detection of Point Mutations.
abstract: The detection of point mutations is required in the diagnosis of many human diseases. The conformal specificity of DNA ligases was elegantly used to distinguish single-nucleotide mismatches. However, to detect point mutations in RNA retroviruses, conventional ligase-mediated approaches require the reverse transcription of viral genomes before separate ligation and amplification steps. We developed one-step ligation on RNA amplification (LRA) for the direct detection of RNA point mutations. The process combines the ligase-mediated joining of two oligonucleotides and subsequent hot start amplification into a single-tube reaction. We report that modifications to the structure of the oligonucleotide ligation probes improve the rate of ligation and the specificity of mutation detection on RNA. We applied LRA to the detection of a common, clinically relevant HIV-1 reverse transcriptase drug-resistant point mutation, K103N, and compared it with allele-specific PCR and pyrosequencing. LRA achieved a limit of specific quantitation of 1:100 (1%), and a limit of specific detection for mutant K103N RNA transcripts among excess wild-type strands of 1:10,000 (0.01%). LRA also exhibited good detection threshold of 5 × 10(2) copies/μL K103N RNA transcripts. LRA is a novel point mutation detection method, with potential utilization in HIV drug resistance detection and early diagnostics of genetic disorders associated with other infectious diseases and cancer.
authors: Center for Biomedical Engineering and the School of Engineering and Medical Sciences, Brown University, Providence, Rhode Island.
authors: Lei
authors: L
authors: Zhang
authors: Center for Biomedical Engineering and the School of Engineering and Medical Sciences, Brown University, Providence, Rhode Island.
authors: Jingjing
authors: J
authors: Wang
authors: Division of Infectious Diseases, Warren Alpert Medical School, Brown University, Providence, Rhode Island.
authors: Mia
authors: M
authors: Coetzer
authors: Center for Biomedical Engineering and the School of Engineering and Medical Sciences, Brown University, Providence, Rhode Island.
authors: Stephanie
authors: S
authors: Angione
authors: Division of Infectious Diseases, Warren Alpert Medical School, Brown University, Providence, Rhode Island.
authors: Rami
authors: R
authors: Kantor
authors: Center for Biomedical Engineering and the School of Engineering and Medical Sciences, Brown University, Providence, Rhode Island. Electronic address: anubhav_tripathi@brown.edu.
authors: Anubhav
authors: A
authors: Tripathi
mesh_terms: D011088:DNA Ligases / Q000235:genetics; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000821:virology; D054303:HIV Reverse Transcriptase / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D008026:Ligation / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D009841:Oligonucleotides / Q000235:genetics; D017354:Point Mutation / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics*
chemical_list: D015345:Oligonucleotide Probes; D009841:Oligonucleotides; D012313:RNA; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase; D011088:DNA Ligases