One step diagnosis by dendron-mediated DNA chip
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Publication Number
US-9023597-B2
Publication Date
2015-05-05
Expiration Date
Abstract
This invention relates to chips containing nucleic acid probes or primers and their use in methods to detect nucleic acid molecules. The invention includes DNA chips in contact with a thermocycler capable of automatically regulating the temperature, temperature cycle times, and number of temperature cycles of the chips to provide genetic diagnosis in one step.
Core Innovation
The invention relates to a dendron-mediated DNA chip system for nucleic acid diagnosis that combines one-step nucleic acid amplification and on-chip hybridization/detection in a sealed chamber. A method is provided in which a nucleic acid molecule is amplified by PCR in the presence of a single stranded nucleic acid probe complementary to the nucleic acid molecule, followed by detection of a double stranded complex attached to a solid surface.
A key structural element is a single stranded nucleic acid probe whose one end is immobilized by attachment to the apex of a conical dendron attached to a solid surface by the base of the conical dendron. After completing the last cycle of PCR, the amplified nucleic acid material is denatured in the presence of the PCR reaction mixture to allow hybridization within the same PCR reaction mixture, thereby forming a double stranded complex in the presence of the immobilized probe and attached conical dendron.
The disclosed chip workflow supports consecutive PCR to hybridization, with or without purification, and detection of the double stranded complex attached to the solid surface. Detection options include fluorescently labeled dNTPs or primers and alternatives such as SYBR Green, electrochemical, and radioisotope detection, with fluorescence scanning on dendron-based chip surfaces.
Claims Coverage
The independent claim is directed to a method for detecting a nucleic acid molecule using PCR with two primers in the presence of a single stranded nucleic acid probe immobilized on a conical dendron, where amplification, denaturation, in-mixture hybridization, and detection occur to yield a double stranded complex attached to the solid surface. Dependent claims further refine the target context and the detection/labeling approach, including labeled primers or nucleotide triphosphates with fluorescent labels, detection of labeled double stranded DNA, mutated sequences, and cDNA generated by reverse transcription of an RNA molecule from the sample.
Dendron-immobilized single stranded probe on conical dendron solid surface
A single stranded nucleic acid probe complementary to the nucleic acid molecule is immobilized via attachment to the apex of a conical dendron attached to a solid surface by the base of the conical dendron.
PCR with probe present followed by in-mixture denaturation and hybridization
The amplified nucleic acid material is produced by PCR, then denatured in the presence of the PCR reaction mixture, and allowed to hybridize to the probe to form a double stranded complex.
Detection of double stranded complex attached to the solid surface
Detecting the double stranded complex attached to the solid surface.
Thermocycler temperature cycling applied to the solid surface
The solid surface is contacted with a thermocycler that regulates temperature, temperature cycle times, and the number of temperature cycles applied to the solid surface.
Labeled amplification and label-based detection
Amplification is performed using a detectable, labeled primer or nucleotide triphosphate incorporated into the amplified nucleic acid, followed by detecting the label.
Fluorescent label detection
The detectable label is a fluorescent label.
Detection using probe complementary to a mutated sequence
Using a mutated nucleic acid sequence and a probe complementary to the mutated sequence.
Use of cDNA generated by reverse transcription
Using a nucleic acid molecule that is cDNA generated by reverse transcription of an RNA molecule from the sample.
Across the independent method and its refinements, the claims are centered on a dendron-immobilized single stranded probe on a conical dendron solid surface and an integrated PCR-to-denaturation-to-hybridization workflow within the same PCR reaction mixture, with detection of the resulting double stranded complex attached to the solid surface. Dependent claims further add thermocycler-controlled temperature cycling at the solid surface and specify detection and labeling options, including fluorescent labels, as well as target refinements such as mutated sequences and cDNA produced by reverse transcription.
Stated Advantages
Supports a one-step nucleic acid diagnosis workflow integrating PCR amplification with subsequent hybridization and detection on-chip in the same PCR reaction mixture.
Enables detection of a double stranded complex attached to a solid surface using an immobilized probe on a conical dendron structure.
Provides detection options including fluorescent detection and alternatives such as SYBR Green, electrochemical, and radioisotope detection.
Documented Applications
Blood-direct PCR using dendron surfaces and fluorescence scanning for nucleic acid diagnosis.
Serum HBV detection using a dendron-mediated DNA chip approach.
One-step RT-PCR using dendron-mediated DNA chip integration of amplification with on-chip hybridization and detection.
p53 codon mutation detection (175/215/216/248) using dendron surfaces and fluorescence scanning.
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