Thermostable Tirt Reverse Transcriptase for High-Temperature cDNA Synthesis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current reverse transcriptase enzymes used in RT-PCR reactions are not stable and effective at high temperatures, leading to issues such as secondary structure formation in RNA templates, non-specific annealing of PCR primers, and reduced fidelity of cDNA synthesis.
Innovation Solution
A thermostable RNA-directed DNA polymerase, Tirt, isolated from Geobacillus stearothermophilus, which retains reverse transcriptase activity up to 75°C, is used for cDNA synthesis and RT-PCR reactions, eliminating secondary structures and improving reaction specificity and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If retroviral reverse transcriptases are used for cDNA synthesis at temperatures below 50°C, then the enzyme activity is optimal, but secondary structures form in the RNA template blocking further processivity
Solution Approach 1:
The patent changes the temperature parameter from below 50°C to 50-75°C to resolve the contradiction. At these elevated temperatures, secondary structures in the RNA template are melted, improving processivity, while the thermostable reverse transcriptase maintains optimal enzyme activity through its thermal stability.
Solution Approach 2:
The patent uses a composite enzyme system combining a thermostable reverse transcriptase (from Thermus thermophilus or Geobacillus stearothermophilus) with RNase H inhibitor. This composite approach allows the enzyme to function effectively at high temperatures while preventing RNA template degradation, resolving both the secondary structure and enzyme stability issues simultaneously.
2Productivity
If cDNA synthesis is performed at temperatures above 50°C, then secondary structures are melted and processivity is improved, but existing reverse transcriptases lose stability and activity
Solution Approach 1:
The patent identifies and utilizes reverse transcriptases from thermophilic organisms (Thermus thermophilus and Geobacillus stearothermophilus) that are naturally adapted to function at 50-75°C. This parameter change in temperature is made possible because the enzyme itself is thermostable, resolving the contradiction between improved processivity and maintained stability.
Solution Approach 2:
The patent creates a functional copy of a thermostable reverse transcriptase enzyme through recombinant DNA technology, cloning the gene from Thermus thermophilus or Geobacillus stearothermophilus into expression vectors. This allows production of the thermostable enzyme in E. coli for use in high-temperature cDNA synthesis reactions.
3Temperature
If DNA-dependent DNA polymerase Tth pol is used for high-temperature cDNA synthesis, then cDNA can be synthesized at high temperatures, but manganese chloride must be added which reduces fidelity of cDNA synthesis
Solution Approach 1:
The patent changes the metal ion parameter from manganese chloride to magnesium chloride in the reaction buffer. This parameter change allows the thermostable reverse transcriptase to maintain high fidelity cDNA synthesis at elevated temperatures without requiring manganese chloride, thus resolving the contradiction between high-temperature synthesis and synthesis fidelity.
4Reliability
If AMV-RT is used for RT-PCR reactions, then RNase H activity is reduced and thermostability is improved, but larger amounts of enzyme and substrate are required and synthesis from long RNA templates is truncated
Solution Approach 1:
The patent changes the enzyme source from AMV-RT to thermostable reverse transcriptases from Thermus thermophilus or Geobacillus stearothermophilus. These enzymes have superior processivity at high temperatures, allowing efficient synthesis from long RNA templates without truncation, while maintaining thermostability through their thermal adaptation.
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
Tirt enables efficient and specific cDNA synthesis at elevated temperatures, reducing non-specific annealing and increasing the length of cDNA products, while maintaining enzyme stability and activity.
Implementation Method 1
a novel isolated polynucleotide sequence from the genome of Bacillus stearothermophilus (Geobacillus stearothermophilus) SEQ ID NO 1, and a novel amino acid sequence (SEQ ID NO 2) encoded by the polynucleotide sequence, the corresponding amino acid sequence comprising a heat-stable protein with reverse transcriptase activity
Implementation Method 2
The invention provides a novel enzyme, Tirt (thermostable intron reverse transcriptase), which has reverse transcriptase activity and retains that activity at temperatures of up to about 75° C.
Data Source
AI summary
The invention relates to an isolated polynucleotide sequence from the genome of Bacillus stearothermophilus (Geobacillus stearothermophilus) and an amino acid sequence encoded by the polynucleotide sequence, the corresponding amino acid sequence comprising a novel enzyme, Tirt (thermostable intron reverse transcriptase), having reverse transcriptase activity and retaining that activity at temperatures of up to about 75° C.