Reverse-Transcriptase DNA Polymerase Mutant for One-Step RNA Amplification
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Solution Overview
Problem
Existing nucleic acid amplification methods face challenges in balancing reverse transcription and nucleic acid amplification reactions in the same reaction solution composition, leading to inefficiencies and risks of cross-contamination, particularly in RT-PCR and RT-isothermal methods.
Innovation Solution
Development of a DNA polymerase mutant with a reverse transcriptase activity, achieved by introducing specific mutations into a 12-amino acid sequence, enhancing its performance in both reverse transcription and nucleic acid amplification reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If a one-step RT-PCR method is used to perform reverse transcription and PCR continuously in the same reaction vessel, then the risk of cross-contamination is reduced, but it becomes difficult to balance the reverse transcription reaction and PCR reaction conditions
Solution Approach 1:
The patent creates a DNA polymerase mutant that performs both reverse transcription and DNA polymerization functions. The mutant enzyme can catalyze reverse transcription of RNA to cDNA and subsequently perform PCR amplification on the synthesized cDNA, all within the same reaction vessel using the same enzyme preparation, thereby eliminating the need for separate reaction steps and reducing cross-contamination risk
2Ease of operation
If the same reaction solution composition is used for both reverse transcription and PCR, then the one-step method can be implemented, but the efficiency of both reactions decreases due to inability to optimize conditions for each reaction type
Solution Approach 1:
The patent introduces specific amino acid mutations (A3 and/or A10 replaced by basic amino acid residues) in the DNA polymerase enzyme to alter its catalytic properties. These parameter changes at the molecular level enable the enzyme to efficiently catalyze both reverse transcription and DNA polymerization reactions under the same reaction conditions, maintaining high productivity while simplifying the操作流程
3Quantity of substance
If conventional DNA polymerase is used for reverse transcription and PCR, then the enzyme maintains high DNA polymerization activity, but the reverse transcription activity is insufficient
Solution Approach 1:
The patent applies local quality modification by introducing specific mutations at particular positions (A3 and/or A10) in the DNA polymerase amino acid sequence. These localized changes confer reverse transcription capability to the enzyme without compromising its overall DNA polymerization function, creating an enzyme with dual functionality and improved reverse transcription efficiency
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The DNA polymerase mutant allows for faster and more sensitive nucleic acid amplification with higher cDNA production, reducing reaction time and improving detection sensitivity compared to conventional methods.
Implementation Method 1
DNA polymerases function intracellularly as an enzyme responsible for DNA synthesis, and polymerize deoxyribonucleoside triphosphates in the presence of metal activators such as Mg2+
Implementation Method 2
an RNA template first needs to be reverse transcribed to cDNA. A typical RT-PCR method is performed by using a reverse transcriptase for synthesizing cDNA from an RNA template
Data Source
AI summary
Provided are: a DNA polymerase mutant having reverse transcriptase activity, the DNA polymerase mutant including a sequence consisting of twelve specific amino acids A1-A12, wherein the DNA polymerase mutant having reverse transcriptase activity is characterized in that the A3 and/or A10 amino acid is substituted by a basic amino acid residue different from that prior to the introduction of mutation; a kit and a composition including the DNA polymerase; a method for producing the DNA polymerase; and a method for modifying an existing DNA polymerase having reverse transcriptase activity.