Modified Taq Polymerase for Rapid Long Polynucleotide Amplification
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Solution Overview
Problem
Conventional methods for amplifying DNA and RNA face limitations such as limited polymerases with reverse transcription activity, contamination issues due to inability to incorporate deoxyuridine triphosphate, and slower amplification speeds, especially for long polynucleotides.
Innovation Solution
Modification of Taq polymerase by replacing arginine at position 651 with glutamic acid and deleting amino acids 1-235 to enhance reverse transcription activity and incorporate deoxyuridine triphosphate, allowing for rapid amplification and detection of long polynucleotides while reducing 5'→3' exonuclease activity to prevent probe degradation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional polymerases are used for amplification, then amplification can be performed, but reverse transcription activity is limited and deoxyuridine triphosphate incorporation is insufficient
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of Taq polymerase, specifically replacing arginine at position 651 with glutamic acid. This biochemical parameter change enables the polymerase to acquire reverse transcription activity and improved deoxyuridine triphosphate incorporation capability, directly resolving the contradiction between versatility and reliability.
Solution Approach 2:
The modified Taq polymerase achieves multi-functionality by combining DNA polymerase activity, reverse transcription activity, and enhanced deoxyuridine triphosphate incorporation in a single enzyme. This universal polymerase can handle both DNA and RNA templates while maintaining contamination prevention capabilities through uracil glycosylase sensitivity.
2Productivity
If amplification speed is increased, then productivity improves, but amplification of long chain polynucleotides becomes more difficult
Solution Approach 1:
The patent achieves high-speed amplification of long chains by modifying the polymerase's amino acid sequence and optimizing reaction conditions. The modified Taq polymerase with glutamic acid at position 651 demonstrates enhanced processivity and speed, achieving at least 20 nucleotides per second while maintaining accuracy for long polynucleotide amplification.
3Productivity
If 5'→3' exonuclease activity is maintained, then primer extension is efficient, but probe degradation occurs during detection
Solution Approach 1:
The patent applies local quality by creating a polymerase variant with modified local properties - the 5'→3' exonuclease activity is reduced or eliminated while retaining 5'→3' polymerase activity. This localized functional modification allows the enzyme to extend primers efficiently without degrading probes, enabling simultaneous amplification and detection.
Solution Approach 2:
The patent segments the enzymatic functions by separating polymerase activity from exonuclease activity in the modified Taq polymerase. This functional segmentation allows the polymerase to perform primer extension while the exonuclease function is suppressed, preventing probe degradation during real-time detection.
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
Enables rapid amplification of long polynucleotides at rates of at least 20 nucleotides per second and prevents contamination, facilitating efficient detection through improved polymerase activity and reduced exonuclease activity, suitable for both DNA and RNA amplification.
Implementation Method 1
a polymerase having amino acid sequence (i) SEQ ID NO:1 wherein the amino acids at positions 1-235 have been deleted (ii) SEQ ID NO:1 or (iii) an amino acid sequence having an amino acid sequence identity of at least 80% to the amino acid sequence of (i) or (ii), and having reverse transcription activity
Implementation Method 2
the polymerase has reverse transcription activity (i.e. reverse transcriptase activity)
Implementation Method 3
its 5'→3' exonuclease activity can be reduced by deleting the amino acids at positions 1-235 in SEQ ID NO:1
Data Source
Figure 1A
Figure 1B
Figure 1C
AI summary
Disclosed is a method of amplifying a polynucleotide, comprising: (a) mixing primers for amplifying the polynucleotide, a polymerase, nucleotide substrates and a template polynucleotide, and (b) amplifying the polynucleotide by a polymerase reaction, wherein the polymerase has an amino acid sequence consisting of SEQ ID NO:1 or an amino acid sequence homologous to SEQ ID NO:1, and wherein the amino acid residue corresponding to position 651 of the amino acid sequence has been replaced with glutamic acid.