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Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences

Title
Fluorescence melting curve analysis using self-quenching dual-labeled peptide nucleic acid probes for simultaneously identifying multiple DNA sequences
Author
황승용
Keywords
Fluorescence melting curve analysis; Peptide nucleic acid; Melt peak; Real-time polymerase chain reaction; High-resolution melting; FMCA; PNA; PCR; HRM
Issue Date
2015-06
Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
Citation
ANALYTICAL BIOCHEMISTRY, v. 484, Page. 143-147
Abstract
Previous fluorescence melting curve analysis (FMCA) used intercalating dyes, and this method has restricted application. Therefore, FMCA methods such as probe-based FMCA and molecular beacons were studied. However, the usual dual-labeled probes do not possess adequate fluorescence quenching ability and sufficient specificity, and molecular beacons with the necessary stem structures are hard to design. Therefore, we have developed a peptide nucleic acid (PNA)-based FMCA method. PNA oligonucleotide can have a much higher melting temperature (T-m) value than DNA. Therefore, short PNA probes can have adequate Tm values for FMCA, and short probes can have higher specificity and accuracy in FMCA. Moreover, dual-labeled PNA probes have self-quenching ability via single-strand base stacking, which makes PNA more favorable. In addition, this method can facilitate simultaneous identification of multiple DNA templates. In conventional real-time polymerase chain reaction (PCR), one fluorescence channel can identify only one DNA template. However, this method uses two fluorescence channels to detect three types of DNA. Experiments were performed with one to three different DNA sequences mixed in a single tube. This method can be used to identify multiple DNA sequences in a single tube with high specificity and high clarity. (C) 2015 Elsevier Inc. All rights reserved.
URI
http://www.sciencedirect.com/science/article/pii/S0003269715002900http://hdl.handle.net/20.500.11754/25684
ISSN
0003-2697; 1096-0309
DOI
10.1016/j.ab.2015.05.022
Appears in Collections:
GRADUATE SCHOOL[S](대학원) > BIONANOTECHNOLOGY(바이오나노학과) > Articles
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