Composition for hot-start reverse transcription reaction or hot-start reverse transcription polymerase chain reaction
Inventors
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Assignees
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BioneerBioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Bioneer is a biotechnology company specializing in molecular biology, offering solutions in nucleic acid synthesis, molecular diagnostics, protein production, and laboratory automation. The company's expertise covers oligonucleotide chemistry, gene synthesis, sample preparation, molecular diagnostics, and analytical services such as mass spectrometry and sequencing, serving clinical, research, and industrial sectors globally.
Publication Number
US-10144972-B2
Publication Date
2018-12-04
Expiration Date
Abstract
A composition for hot-start reverse transcription reaction and a composition for reverse transcription PCR are disclosed. The composition is obtained by adding pyrophosphate and pyrophosphatase to an aqueous solution containing reaction buffer solution, MgCl2, four kinds of dNTPs, and reverse transcription polymerase in a single reaction tube. The composition for hot-start reverse transcription reaction is obtained by freezing or drying the composition. The composition show increased stability and long-term storage stability. Also, disclosed is a composition that additionally includes DNA polymerase, and, thus, enables a hot-start reverse transcription reaction and a PCR reaction to be sequentially performed. A method for amplifying a nucleic acid by using the composition. The composition of the invention can be conveniently and effectively used in multiplex reverse transcription PCRs or real-time quantitative reverse transcription PCR.
Core Innovation
The invention provides hot-start reverse transcription/transcription-PCR compositions in a single reaction tube that include Mg2+, four kinds of dNTPs, a reverse transcription polymerase, pyrophosphate (PPi), and a pyrophosphatase (PPase). A pyrophosphate/pyrophosphatase system is included to inhibit non-specific reverse transcriptions of additional RNAs present in the sample and to reduce mispriming and non-specific amplification by hydrolyzing PPi during reaction conditions.
When used with primer-hybridization temperatures, the system reactivates reverse transcription by removing the PPi-mediated inhibition. The invention further includes compositions that are frozen or dried to improve stability and enable long-term storage.
In addition, cancer detection is supported by providing a template RNA with a nucleic acid polymerization terminator bound to its 3′ end to prevent non-specific nucleic acid polymerization, thereby enabling improved amplification and detection performance, including enhanced detection limit for a cancer marker.
Claims Coverage
The document provides two independent claims. The first claim covers single-tube reverse transcription using an Mg2+ plus PPi/PPase hot-start composition to inhibit non-specific reverse transcription of additional RNAs while producing cDNA of a template RNA. The second claim covers single-tube cancer-cell detection using a template RNA with a 3′-end nucleic acid polymerization terminator and PPi/PPase with a biomarker amplification primer, followed by analyzing an amplification product.
Single-tube hot-start reverse transcription with PPi/PPase inhibition
A method of reverse transcription in which a sample is mixed with a composition in a single reaction tube comprising an Mg2+ ion, four kinds of dNTPs, a reverse transcription polymerase, pyrophosphate, and a pyrophosphatase, wherein the sample further comprises additional RNAs other than the template RNA, and wherein the method inhibits non-specific reverse transcriptions of the additional RNAs to provide a complementary DNA of the template RNA.
Single-tube cancer detection using template RNA with 3′-end polymerization terminator and PPi/PPase
A method for detecting a cancer cell by mixing a sample in a single reaction tube with a composition comprising a template RNA having a nucleic acid polymerization terminator bound to the 3′ end to prevent non-specific nucleic acid polymerization, an Mg2+ ion, four kinds of dNTPs, a reverse transcription polymerase, nucleic acids, a primer for synthesizing cDNA of the template RNA, a pyrophosphate, a pyrophosphatase, and a primer which binds and amplifies a nucleic acid of a biomarker of the cancer cell; performing reverse transcription and amplification to provide an amplification product; and analyzing the amplification product to detect the cancer cell.
Overall, the claims center on single-reaction-tube hot-start reverse transcription using an Mg2+ plus PPi/PPase composition to inhibit non-specific reverse transcription of additional RNAs, and on cancer detection using a template RNA with a 3′-end nucleic acid polymerization terminator together with PPi/PPase and a biomarker amplification primer, followed by analysis of the amplification product.
Stated Advantages
Inhibits non-specific reverse transcriptions of additional RNAs.
Reduces mispriming and non-specific amplification.
Improves detection performance, including enhanced detection limit for a cancer marker (KRT8) when used in combination with nucleic-acid-like 3′ termination.
Enables improved stability and long-term storage by using frozen or dried compositions.
Supports reduced multiplex background and compatibility with real-time RT-PCR.
Documented Applications
Detection of hepatitis C virus (HCV) RNA by hepatitis C virus RT-PCR using the hot-start reverse transcription system with PPi/PPase.
Multiplex reverse transcription PCR with reduced multiplex background using the PPi/PPase hot-start approach.
Real-time quantitative RT-PCR using the PPi/PPase hot-start composition.
Detection of a cancer marker (KRT8) in a cancer assay using a template RNA with a nucleic acid polymerization terminator bound to the 3′ end.
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