Sso7 Polymerase Conjugate Mutations Reduce Non-Specific Amplification
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Solution Overview
Problem
Current nucleic acid amplification reactions, such as PCR, face challenges in reducing non-specific amplification activity, which can lead to unwanted product formation and decreased specificity.
Innovation Solution
The development of an Sso7 polymerase conjugate protein, where an amino acid at position K28 of the Sso7 domain is altered from lysine to alternative amino acids like serine, threonine, or cysteine, reducing non-specific amplification activity by at least 10% compared to a control conjugate protein, and is combined with a thermally stable polymerase domain for improved processivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of moving object
If an Sso7 domain is joined to a polymerase to improve processivity, then the enzyme exhibits increased processivity, but non-specific amplification activity increases
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the Sso7 domain (positions 28, 43, and 53) to alter the enzyme's binding characteristics. These parameter changes at the molecular level reduce non-specific DNA binding while preserving processivity, directly resolving the technical contradiction between improved processivity and reduced non-specific activity.
Solution Approach 2:
The patent applies local quality by making targeted modifications at specific locations (amino acid positions 28, 43, and 53) within the Sso7 domain rather than uniformly modifying the entire protein. This localized approach allows the enzyme to maintain high processivity in the DNA-binding regions while reducing non-specific activity at particular interaction sites.
2Duration of action of moving object
If the Sso7 domain binds non-specifically to double-stranded nucleic acid, then processivity is improved, but specificity of amplification decreases
Solution Approach 1:
The patent changes the chemical parameters of the Sso7 domain by substituting amino acids at positions 28, 43, and 53 with residues that have different binding affinities and characteristics. This alters the enzyme's interaction parameters with double-stranded nucleic acid, reducing non-specific binding while maintaining template-dependent processivity, thereby improving amplification specificity.
Solution Approach 2:
The patent converts the harmful non-specific binding property into a benefit by strategically modifying the Sso7 domain to reduce non-specific interactions. The mutated enzyme maintains the advantageous processivity enhancement while eliminating the harmful non-specific amplification, effectively converting a dual-natured property into a purely beneficial one.
Data Source
Figure 1
AI summary
Improved Sso7-polymerase conjugate proteins are provided. The present invention provides for an Ss07 polymerase conjugate protein comprising an Ss07 domain linked to a polymerase; wherein: an amino acid of the Ss07 domain corresponding to K28 of SEQ 10 NO:2 is not lysine (K); and/or an amino acid of the Ss07 domain corresponding to R43 of SEQ ID NO:2 is not arginine (R), wherein the conjugate protein has a decreased non-specific amplification activity compared to an otherwise identical control conjugate protein in which the amino acid of the Ss07 domain corresponding to K28 of SEQ ID NO:2 is lysine (K) and the amino acid of the Ss07 domain corresponding to R43 of SEQ ID NO:2 is arginine (R).