Ligase-Assisted DNA Circularization for Fragmented Genome Amplification
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Solution Overview
Problem
Current methods for amplifying short, fragmented DNA sequences are inefficient, leading to decreased amplification speed, significant sequence dropout, and sequence bias, especially when using conventional whole-genome amplification techniques like multiple displacement amplification (MDA) or ligation-mediated PCR, which require intermediate isolation and purification steps and often result in inadequate genome coverage.
Innovation Solution
A method for generating single-stranded DNA circles from linear DNA by end-repairing with a polynucleotide kinase and performing intra-molecular ligation with a ligase in a single reaction vessel, without intervening isolation or purification steps, using a pre-adenylated ligase or a non-adenylated ligase capable of template-independent ligation, allowing for efficient circularization and amplification of short DNA sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional whole-genome amplification techniques like multiple displacement amplification (MDA) are used, then amplification of high molecular weight target DNA is achieved, but amplification speed decreases and sequence dropout increases when target DNA is short and fragmented
Solution Approach 1:
The invention changes the fundamental amplification parameters by using isothermal conditions instead of thermal cycling, and by using strand-displacement polymerase to displace hybridized strands without denaturation. This allows rapid amplification of short fragmented DNA while maintaining comprehensive genome coverage, resolving the contradiction between amplification speed and coverage for fragmented DNA targets
2Productivity
If ligation-mediated PCR is used to amplify fragmented dsDNA, then amplification is achieved, but only a small fraction of fragmented DNA gets amplified leading to inadequate genome coverage
Solution Approach 1:
The invention extracts and eliminates the ligation step from the amplification process. By using strand-displacement polymerase that can directly amplify fragmented DNA without requiring ligation to form concatemers, the method achieves both high amplification efficiency and comprehensive genome coverage, resolving the contradiction between productivity and reliability
3Reliability
If double-stranded DNA ends are repaired and blunt-end ligated to form circles, then circularization is achieved, but DNA fragments less than 500 bp in length are difficult to circularize
Solution Approach 1:
The invention inverts the conventional approach by not requiring circularization at all. Instead, it uses linear fragmented DNA directly as templates for strand-displacement amplification. This inversion eliminates the circularization efficiency bottleneck for short fragments while maintaining the ability to amplify comprehensive genomic coverage
4Reliability
If intermediate isolation and purification steps are included in the workflow, then ligation and amplification can be performed sequentially, but the workflow becomes cumbersome and time-consuming
Solution Approach 1:
The invention merges the amplification step with the template preparation into a single unified reaction. By using strand-displacement polymerase that can directly use linear fragmented DNA as templates, the method combines what were previously separate steps (template preparation and amplification) into one simultaneous process, eliminating intermediate purification steps while maintaining reaction accuracy
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
This approach enables balanced and sensitive DNA amplification with reduced sequence dropout and bias, preferentially amplifying fragmented DNA over high molecular weight genomic DNA, improving amplification speed and coverage, and simplifying the workflow by eliminating the need for intermediate purification steps.
Implementation Method 1
performing an intra-molecular ligation of the repaired, ligatable DNA sequence with a ligase in order to generate the single-stranded DNA circle
Implementation Method 2
end-repairing the linear DNA by incubating it with a polynucleotide kinase in the presence of a phosphate donor to generate a ligatable DNA sequence having a phosphate group at a 5' terminal end
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
Provided herein are methods for generation and amplification of a single-stranded DNA circle in a single reaction vessel from a linear DNA without any intervening purification steps. The single-stranded DNA circle is generated via a template-independent single-stranded DNA ligation. Whole-genome amplification of circulating nucleic acids extracted from blood is provided. Kits for performing the disclosed methods are also provided.