Method of quantitatively and qualitatively analyzing biomaterial in real-time
Inventors
Kim, Lee-Kyung • Paek, Mun-Cheol • Ku, Su-Jin • Park, Sun-Young • Lee, Do-Bu • Park, Jae-Hyung • Lee, Ki-Chang • Kim, Nam-Joong
Assignees
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Publication Number
US-10364459-B2
Publication Date
2019-07-30
Expiration Date
Abstract
A method of quantitatively and qualitatively analyzing a biomaterial in real-time, the method comprising preparing a device for detecting a biomaterial, feeding a complex of first and second probes, a forward primer, a reverse primer, a sample comprising deoxynucleotide triphosphate, a polymerase having exonuclease activity, and a sample comprising target genes, and a reaction solution comprising a buffer into the reaction container, performing polymerase chain reaction comprising denaturation of the target genes in the sample, hybridization of the target genes, the complex, and the forward and reverse primers in the sample, and elongation of the primers through the polymerase having exonuclease activity, allowing for elongation of the second probe on the third probe by the polymerase after hybridizing the released second probe and the third probe fixed to the biochip, detecting a first fluorescence signal by the first phosphor and a second fluorescence signal by the second phosphor.
Core Innovation
The invention provides a device and a method for quantitatively and qualitatively analyzing a biomaterial in real-time by combining polymerase chain reaction and DNA microarray with a workflow that generates and detects fluorescence signals without requiring a separately opened reaction vessel. A reaction container comprises an opening at an upper part thereof, and an element part is separately connected to the reaction container via the opening, wherein the element part comprises a cap coupled to the opening of the reaction container. A rod extends from a lower part of the cap and carries a biochip having a third probe fixed to a surface of the biochip.
A complex of a first probe and a second probe, together with a forward primer, a reverse primer, a polymerase having exonuclease activity, deoxynucleotide triphosphate (dNTP), and a sample comprising target genes is placed into the reaction container with a buffer as a reaction solution. The first probe comprises oligonucleotide sequences complementary to nucleic acid sequences of the target genes and a first phosphor for generating a first fluorescence signal together with a first quencher for quenching the first phosphor, while the second probe comprises oligonucleotide sequences complementary to the third probe and is configured to be disassembled and released from a complex hybridized with the target genes by exonuclease activity during primer elongation.
During the polymerase chain reaction, denaturation, hybridization only between the target genes and the first probe of the complex, and elongation through the polymerase with exonuclease activity are performed, such that when the polymerase reaches the complex hybridized with the target genes, the second probe and the first phosphor are disassembled and released. After allowing DNA microarray, elongation of the released second probe on the third probe fixed to the biochip is performed, where the third probe comprises a second phosphor and a second quencher, and as the second probe is elongated on the third probe the secondary structure of the third probe is disassembled so that the second quencher is spaced from the second phosphor to generate a second fluorescence signal. The method detects both the first fluorescence signal by the first phosphor and the second fluorescence signal by the second phosphor.
Claims Coverage
The document contains at least one independent claim (clm-00001) describing a real-time method that integrates a PCR reaction with a DNA microarray on a biochip to produce and detect two fluorescence signals using probe disassembly/release and subsequent hybridization/elongation on a third probe fixed to the biochip. The inventive features are centered on a device configuration with a reaction container and a rod-mounted biochip and a two-fluorophor signaling mechanism using exonuclease-driven probe disassembly followed by DNA microarray elongation and fluorescence generation.
Biochip on a rod coupled via an element part through an opening
Preparing a device in which the reaction container has an opening at an upper part, and an element part is separately connected to the reaction container via the opening, the element part including a cap coupled to the opening and a rod extended from a lower part of the cap, wherein the rod comprises a biochip having a third probe fixed to a surface of the biochip.
First probe complex with first phosphor/quencher and exonuclease-disassembled second probe
Feeding into the reaction container a complex of first and second probes, a forward primer, a reverse primer, dNTP, a polymerase having exonuclease activity, and a sample comprising target genes and buffer, wherein the first probe includes oligonucleotide sequences complementary to target genes and includes a first phosphor and a first quencher, and wherein the second probe does not include sequences complementary to the target genes but includes oligonucleotide sequences complementary to the third probe.
Exonuclease activity causes disassembly/release during PCR to generate first fluorescence
Performing polymerase chain reaction comprising denaturation of the target genes, hybridization only between the target genes and the first probe of the complex, and elongation of the primers through the polymerase having exonuclease activity, wherein when the polymerase reaches the complex hybridized with the target genes during elongation, the second probe and the first phosphor are disassembled and released from the complex hybridized with the target genes by the exonuclease activity, and a first fluorescence signal is generated by the first phosphor.
DNA microarray with elongation on the third probe to generate second fluorescence
Allowing DNA microarray in which elongation of the second probe on the third probe by the polymerase after hybridizing the released second probe and the third probe fixed to the biochip are performed, wherein the third probe comprises a second phosphor for generating a second fluorescence signal and a second quencher for quenching the second phosphor, and as the second probe is elongated on the third probe the secondary structure of the third probe is disassembled, and the second quencher is spaced from the second phosphor, and a second fluorescence signal is generated by the second phosphor.
Detect first and second fluorescence signals in real time
Detecting a first fluorescence signal by the first phosphor and a second fluorescence signal by the second phosphor as part of the real-time quantitative and qualitative analysis.
Across the independent claim, the core inventive aspects are the device arrangement with a rod-mounted biochip incorporated via a capped opening on a reaction container, and a fluorescence generation mechanism where exonuclease activity disassembles and releases a second probe and first phosphor during PCR to produce a first fluorescence signal, followed by DNA microarray elongation on a third probe carrying a second phosphor/quencher to produce a second fluorescence signal that is detected alongside the first.
Stated Advantages
Real-time quantitative and qualitative analysis of a biomaterial.
Generates a first fluorescence signal by the first phosphor and a second fluorescence signal by the second phosphor for detection during the integrated workflow.
Reduces the need for multiple tubes, scanners, opening, and washing, as stated in the provided content.
Documented Applications
No documented applications found
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