Modified Taq Polymerase for Enhanced Reverse Transcription and Long cDNA Synthesis
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Solution Overview
Problem
Current DNA polymerases used in PCR lack convenience, reliability, and versatility for amplifying various DNA templates, with limitations in reverse transcription activity and primer extension efficiency, particularly for long cDNAs and RNA templates that form complex structures.
Innovation Solution
Development of novel DNA polymerases by modifying the Taq polymerase sequence with specific amino acid insertions and substitutions, such as the insertion of -A737-A738-A739-A740-A741-A742-A743-A744-A745- between positions 736 and 737, and mutations at positions 117, 119, 142, and 144, to enhance reverse transcription activity and primer extension efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Taq DNA polymerase is used for PCR amplification, then the amplification can be performed at high temperatures, but the reverse transcription activity is weak and primer extension efficiency is limited
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of Taq DNA polymerase through site-directed mutagenesis. Specific amino acid residues (positions 737-745) were changed to alter the enzyme's catalytic properties, resulting in enhanced reverse transcription activity and primer extension efficiency while maintaining thermostability
Solution Approach 2:
The patent creates a composite enzyme system by combining modified Taq DNA polymerase with specific molecular structures. The chimeric polymerase incorporates domains from different sources (Taq polymerase and other thermostable polymerases), creating a hybrid enzyme with superior functionality for both reverse transcription and DNA amplification
2Reliability
If Tth DNA polymerase is used for reverse transcription, then the reaction can be performed at high temperatures with strong reverse transcription activity, but the enzyme is not suitable for synthesizing long cDNAs
Solution Approach 1:
The patent modifies the amino acid sequence parameters of the polymerase enzyme to enhance its ability to synthesize long cDNAs. By changing specific residues in the catalytic domain and extending the enzyme's structure, the modified polymerase achieves both high reverse transcription activity and the capability to synthesize long cDNA products
Solution Approach 2:
The patent divides the polymerase enzyme into functional domains and selectively modifies them. The N-terminal domain retains reverse transcription activity while the C-terminal domain is extended to enhance processivity for long cDNA synthesis, creating a segmented optimization strategy
3Measurement precision
If Family A DNA polymerases are used for nucleotide sequencing, then the recognition of dideoxynucleotides is superior, but the enzymes produce more errors during DNA amplification
Solution Approach 1:
The patent applies local quality by making site-specific modifications to the polymerase enzyme. The active site region is optimized for dideoxynucleotide recognition while other regions are modified to reduce error rates during amplification, creating different functional zones with specialized properties within the same enzyme
Data Source
AI summary
Provided are various novel DNA polymerases. Provided is a DNA polymerase comprising an amino acid sequence modified from the amino acid sequence of SEQ ID NO: 8 by inserting nine amino acids “-A737-A738-A739-A740-A741-A742-A743-A744-A745-” between the amino acid residue at position 736 and the amino acid residue at position 737,wherein:A737 is an amino acid residue having a non-polar aliphatic side chain;A738 is an amino acid residue having a non-polar aliphatic side chain;A739 is an amino acid residue having a positively charged side chain;A740 is an amino acid residue having a positively charged side chain;A741 is an amino acid residue having a non-polar aliphatic side chain;A742 is an amino acid residue having a non-polar aliphatic side chain;A743 is any given amino acid residue;A744 is an amino acid residue having a positively charged side chain; andA745 is an amino acid residue having a non-polar aliphatic side chain).