Modified Klenow Fragment for Longer Sequencing Reads
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Solution Overview
Problem
The wild-type Klenow and exo-Klenow fragments exhibit poor performance in nucleic acid sequence amplification, particularly in sequencing, with suboptimal sequencing average read length and effective data measures, and are not widely used in commercial amplification platforms due to low temperature resistance and polymerization capacity.
Innovation Solution
A modified Klenow fragment with specific amino acid substitutions at positions F762, A842, 1709, and P603, or functionally equivalent positions, enhancing DNA polymerase activity, affinity for substrates, and compatibility with modified nucleotides, leading to longer read lengths and higher quality data in sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If wild-type or exo-Klenow fragment is used for nucleic acid amplification, then the temperature control requirement is low and molecular weight is small, but the polymerization capacity is low and sequencing performance is poor
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions at positions F762, A842, I709, and P603 in the Klenow fragment sequence. These sequence parameter changes fundamentally alter the enzyme's properties, transforming it from low polymerization capacity to high polymerization capacity with improved sequencing performance while maintaining ease of operation at lower temperatures
2Device complexity
If wild-type or exo-Klenow fragment is used for sequencing, then the enzyme structure is simple, but the sequencing average read length is short and effective data amount is low
Solution Approach 1:
The patent changes the sequence parameters of the Klenow fragment by introducing specific amino acid substitutions at positions F762, A842, I709, and P603. These parameter changes enhance the enzyme's affinity for substrates and compatibility with modified nucleotides, directly improving sequencing read length and effective data amount while maintaining relatively simple enzyme structure
3Reliability
If commercially available Klenow fragment is used, then it shows better performance than wild-type, but still lacks resistance to high temperature and has limited polymerization capacity
Solution Approach 1:
The patent applies parameter changes through specific amino acid substitutions at positions F762, A842, I709, and P603 that simultaneously improve sequencing performance and enhance temperature resistance. The modified enzyme maintains high reliability in sequencing while gaining improved thermal stability compared to commercially available variants
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 modified Klenow fragment demonstrates significantly improved DNA polymerase activity, resulting in increased sequencing read lengths and reduced base repeat ratios, outperforming commercial enzymes under specific conditions, and is suitable for use in nucleic acid sequencing and DNA end-filling.
Implementation Method 1
The Klenow fragment retains the 5'-3' polymerase activity and the 3'-5' exonuclease activity of DNA polymerase I... The modified Klenow fragment has high synthesis capacity, including high synthesis speed and/or high affinity for a substrate
Data Source
AI summary
Provided are a modified Klenow fragment and an application thereof; specifically provided is a modified Klenow fragment, wherein at least one position or functionally equivalent position among F762, A842, 1709 and P603 in the amino acid sequence of the modified Klenow fragment contains at least one amino acid substitution mutation. The modified Klenow fragment has higher DNA polymerase activity than a wild-type Klenow fragment and can be applied to sequencing.