Isothermal target and signaling probe amplification method, based on a combination of an isothermal chain amplification technique and a fluorescence resonance energy transfer cycling probe technology.
An iTPA (isothermal target and signaling probe amplification) method for the quantitative detection of nucleic acids, based on a combination of novel ICA (isothermal chain amplification) and fluorescence resonance energy transfer cycling probe technology (FRET CPT), is described. In the new ICA method, which relies on the strand displacement activity of DNA polymerase and the RNA degrading activity of RNase H, two displacement events occur in the presence of four specially designed primers. This phenomenon leads to powerful amplification of target DNA. Since the amplification is initiated only after hybridization of the four primers, the ICA method leads to high specificity for the target sequence. As part of the new ICA method, iTPA is achieved by incorporating FRET CPT to generate multiple fluorescence signals from a single target molecule. Using the resulting dual target and signaling probe amplification system, even a single copy level of a target gene can be successfully detected and quantified under isothermal conditions.
Jung
Cheulhee
C
Department of Chemical and Biomolecular Engineering, KAIST, 335 Gwahak-ro, Yuseong-gu, Daejeon, Republic of Korea.
Chung
Ji Won
JW
Kim
Un Ok
UO
Kim
Min Hwan
MH
Park
Hyun Gyu
HG
D004247:DNA / Q000737:chemistry*; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D015894:Genome, Human; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D016914:Ribonuclease H / Q000378:metabolism
D017931:DNA Primers; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H