Method for detecting nucleic acids by promoting branched DNA complex formation
Inventors
Chae, Chi-Bom • Kim, Kyung-tae • Kang, Jong Hun
Assignees
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Publication Number
US-9868983-B2
Publication Date
2018-01-16
Expiration Date
Abstract
Disclosed is a method for detecting nucleic acids by promoting branched DNA complex formation. The target nucleic acid detection signal and sensitivity can be dramatically increased by promoting self assembly of branched DNA between a plurality of amplified DNA targets and a single-chain oligonucleotide probe, by means of the integrated implementation of PCR, thermal denaturation and hybridization in a single reaction mixture.
Core Innovation
The invention provides a method for detecting a target nucleic acid molecule by performing an asymmetric polymerase chain reaction of a single reaction mixture. The single reaction mixture comprises a target nucleic acid molecule to be detected, a labeled forward primer, and a labeled reverse primer, wherein the labeled reverse primer is used at a concentration higher than that of the forward primer. A nucleic acid probe attached via a linker molecule at its 5′ end to a solid support is present, and the nucleic acid probe comprises a sequence entirely or partially complementary to the target nucleic acid molecule.
After asymmetric polymerase chain reaction, the amplified nucleic acid molecules are thermally denatured in the presence of the nucleic acid probe attached to the solid support. The denatured amplified nucleic acid molecules are hybridized to the nucleic acid probe at a temperature from 50° C. to 60° C., where hybridization of multiple amplified nucleic acid molecules to the probe promotes formation of a branched nucleic acid complex on the solid support. The branched nucleic acid complex is then detected.
In the integrated approach, the method delivers the whole reaction mixture to a solid support surface that includes a nucleic acid probe attached via a linker molecule at the 5′ end, and the method thermally denatures the whole reaction mixture before or after delivering it to the solid support. Hybridizing multiple amplified nucleic acid molecules denatured in this way with the probe at 50° C. to 60° C. again promotes formation of a branched nucleic acid complex, followed by detecting the hybridized branched nucleic acid complex.
Claims Coverage
The document contains two independent method claims that share the same core inventive concept: integrated asymmetric PCR with probe-driven formation of a branched nucleic acid complex on a solid support, followed by detection. Across these independent claims, the inventive features revolve around labeled primers with an imbalanced primer concentration, a solid-support-attached probe linked at the 5′ end, thermal denaturation and hybridization at 50° C. to 60° C. to promote branched nucleic acid complex formation, and detection of the hybridized branched nucleic acid complex.
Asymmetric PCR with labeled primer concentration imbalance in a single reaction mixture
Performing an asymmetric polymerase chain reaction of a single reaction mixture comprising a target nucleic acid molecule to be detected, a labeled forward primer and a labeled reverse primer, wherein the labeled reverse primer is used at a concentration higher than that of the forward primer, in the presence of a nucleic acid probe attached via a linker molecule at its 5′ end to a solid support.
Thermal denaturation and probe hybridization to promote branched nucleic acid complex formation
Thermally denaturing the amplified nucleic acid molecules in the presence of the nucleic acid probe attached to the solid support and hybridizing the denatured amplified nucleic acid molecules to the nucleic acid probe at a temperature from 50° C. to 60° C., wherein the hybridization of multiple amplified nucleic acid molecules to said probe promotes formation of a branched nucleic acid complex on the solid support.
Detection of the hybridized branched nucleic acid complex on the solid support
Detecting the hybridized branched nucleic acid complex.
Delivering the whole reaction mixture to a solid support bearing a 5′-linked complementary probe
Amplifying a target nucleic acid molecule using an asymmetric PCR using a labeled forward primer and a labeled reverse primer, wherein the labeled reverse primer is used at a concentration higher than that of the forward primer, and delivering the whole reaction mixture to a solid support surface which includes a nucleic acid probe attached via a linker molecule at the 5′ end and comprising a sequence entirely or partially complementary to the target nucleic acid molecule.
Thermal denaturation before or after delivery and hybridization at 50° C. to 60° C. to form the branched complex
Thermally denaturing the whole reaction mixture before or after the reaction mixture is delivered to the solid support surface; and hybridizing multiple amplified nucleic acid molecules denatured in this way with the nucleic acid probe attached to the solid support at a temperature from 50° C. to 60° C. to promote formation of a branched nucleic acid complex.
Detecting the hybridized branched nucleic acid complex after integrated processing
Detecting the hybridized branched nucleic acid complex.
The claim coverage is centered on integrated asymmetric PCR combined with probe-driven branched nucleic acid complex formation on a solid support. Both independent claims require labeled forward and reverse primers with a higher concentration of the labeled reverse primer, a nucleic acid probe attached via a 5′ linker to a solid support, thermal denaturation, hybridization at 50° C. to 60° C. to promote formation of a branched nucleic acid complex, and detection.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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