Using force spectroscopy analysis to improve the properties of the hairpin probe.
The sensitivity of hairpin-probe-based fluorescence resonance energy transfer (FRET) analysis was sequence-dependent in detecting single base mismatches with different positions and identities. In this paper, the relationship between the sequence-dependent effect and the discrimination sensitivity of a single base mismatch was systematically investigated by fluorescence analysis and force spectroscopy analysis. The same hairpin probe was used. The uneven fluorescence analysis sensitivity was obviously influenced by the guanine-cytosine (GC) contents as well as the location of the mismatched base. However, we found that force spectroscopy analysis distinguished itself, displaying a high and even sensitivity in detecting differently mismatched targets. This could therefore be an alternative and novel way to minimize the sequence-dependent effect of the hairpin probe. The advantage offered by force spectroscopy analysis could mainly be attributed to the percentage of rupture force reduction, which could be directly and dramatically influenced by the percentage of secondary structure disruption contributed by each mismatched base pair, regardless of its location and identity. This yes-or-no detection mechanism should both contribute to a comprehensive understanding of the sensitivity source of different mutation analyses and extend the application range of hairpin probes.
Yang
Liu
L
State Key Laboratory of Chemo/Biosensing and Chemometrics, Biomedical Engineering Center, Engineering Research Center for Bio-Nanotechnology of Hunan Province, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China
Wang
Kemin
K
Tan
Weihong
W
Li
Huimin
H
Yang
Xiaohai
X
Ma
Changbei
C
Tang
Hongxing
H
D020137:Base Pair Mismatch; D004247:DNA / Q000737:chemistry; D004252:DNA Mutational Analysis / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D018625:Microscopy, Atomic Force; D009691:Nucleic Acid Denaturation; D015345:Oligonucleotide Probes / Q000737:chemistry*; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D015345:Oligonucleotide Probes; D004247:DNA