Ligase-Assisted DNA Circularization for Fragmented Genome Amplification
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Solution Overview
Problem
Current methods for whole-genome amplification of short, fragmented DNA sequences are inefficient, leading to decreased amplification speed, significant sequence dropout, and sequence-biased amplification, especially when the target DNA is short or highly fragmented, and require multiple intervening isolation and purification steps, which can result in DNA loss and inhibition of subsequent amplification reactions.
Innovation Solution
A method involving template-independent single-stranded DNA ligation to generate a single-stranded DNA circle followed by rolling circle amplification using a random primer mixture in a single reaction vessel, without any intervening isolation or purification steps, using a ligase capable of intra-molecular ligation and a random primer mixture with nucleotide analogues to enhance amplification efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional whole-genome amplification methods are used on short, fragmented DNA, then amplification can be attempted, but amplification speed decreases and sequence dropout increases
Solution Approach 1:
The patent changes the fundamental parameters of the amplification approach by using template-independent ligation to circularize ssDNA fragments, followed by rolling circle amplification. This transforms the amplification mechanism from conventional PCR/MDA to a circular template-based system, enabling efficient amplification of short fragmented DNA with reduced sequence dropout and bias.
Solution Approach 2:
The patent introduces an intermediary step of template-independent ligation to convert linear ssDNA fragments into circular templates. This intermediary transformation enables the subsequent rolling circle amplification to proceed efficiently, overcoming the limitations of direct amplification of fragmented DNA.
2Ease of manufacture
If multiple isolation and purification steps are performed between ligation and amplification, then reaction components can be separated, but DNA loss occurs and subsequent amplification is inhibited
Solution Approach 1:
The patent merges the ligation and amplification reactions into a single continuous process in the same reaction vessel. By eliminating intermediate purification steps, the method prevents DNA loss and avoids inhibition of amplification that would otherwise occur during separate processing steps.
Solution Approach 2:
The patent maintains continuous useful action by performing ligation and amplification without interruption or intermediate handling. The reaction proceeds continuously from template-independent ligation of ssDNA fragments to rolling circle amplification of the circularized products, maximizing DNA recovery and minimizing inhibition.
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 efficient, balanced, and sensitive amplification of short DNA sequences with reduced sequence dropout and amplification bias, maintaining representative genome information and overcoming inhibition issues, allowing for effective amplification of even highly fragmented DNA in a single reaction vessel.
Implementation Method 1
template-independent single-stranded DNA ligation to generate a single-stranded DNA circle
Implementation Method 2
The complementary strand is assembled from deoxynucleoside triphosphates (dNTPs) by a DNA polymerase. The complementary strand synthesis proceeds in 5′→3′ direction starting from the 3′ terminal end of a primer sequence
Implementation Method 3
The double-stranded DNA may be denatured to produce single-stranded DNA (ssDNA)
Implementation Method 4
amplifying the single-stranded DNA circle via rolling circle amplification
Data Source
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 linear chromosomal DNA in a single tube using ligation-assisted DNA amplification is also provided.