Colocalization Complex Modulates Nucleic Acid Library Bias
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Solution Overview
Problem
DNA library amplification methods, such as PCR and rolling circle amplification, suffer from amplification bias, where different DNA sequences are amplified with varying efficiencies, leading to altered representation and loss of diversity in the library over cycles.
Innovation Solution
The use of a DNA binding protein and a guide RNA, specifically self-targeting guide RNAs (stgRNAs), to form a colocalization complex that modulates amplicons by binding and enzymatically acting on the most abundant amplicons, reducing their abundance and reversing amplification bias.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If DNA library amplification is performed using PCR or rolling circle amplification, then the number of copies available from DNA molecules is increased, but amplification bias occurs where different sequences are amplified with variable efficiencies leading to altered representation and loss of diversity
Solution Approach 1:
The patent introduces an intermediary substance (the modulating agent) that selectively binds to abundant amplicons to suppress their over-amplification. This intermediary acts as a mediator between the amplification process and the bias problem, allowing the system to achieve both high copy numbers and accurate representation by preventing preferential amplification of certain sequences
Solution Approach 2:
The patent implements a feedback mechanism where the amplification process itself generates information about sequence abundance, and this information is used to modulate subsequent amplification cycles. The system monitors which sequences are being over-amplified and automatically adjusts amplification efficiency through the modulating agent, creating a self-regulating system that maintains representation accuracy while increasing copy numbers
2Productivity
If multiple cycles of amplification are performed to increase library size, then more copies are produced, but amplification bias accumulates and diversity is lost
Solution Approach 1:
The patent applies the modulating agent continuously throughout the amplification process rather than as a single intervention. This continuous action ensures that amplification bias is suppressed throughout all cycles, maintaining library diversity even as productivity increases. The modulating agent remains active throughout the entire amplification process to prevent bias accumulation
Solution Approach 2:
The modulating agent serves as a continuous intermediary that mediates between the amplification machinery and the template sequences throughout multiple cycles. This intermediary prevents the accumulation of bias by continuously suppressing over-amplification of abundant sequences, allowing productivity to increase while preserving the stable composition and diversity of the library
3Measurement precision
If amplification is performed to increase available copies, then sensitivity is improved, but bias in sequence representation increases
Solution Approach 1:
The modulating agent acts as an intermediary that allows the system to achieve high detection sensitivity through amplification while preventing information loss about sequence representation. By selectively suppressing over-amplified sequences, the intermediary ensures that the amplified library maintains accurate representation information even at high copy numbers, enabling both sensitive detection and accurate measurement
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 effectively reduces the abundance of the most abundant amplicons, thereby correcting amplification bias and maintaining library diversity, ensuring a more representative DNA library.
Implementation Method 1
a guide RNA, specifically self-targeting guide RNAs (stgRNAs), to form a colocalization complex that modulates amplicons by binding
Data Source
AI summary
A method of processing a collection of nucleic acid sequences is provided including connecting an adaptor to one or more or each nucleic acid sequence in the collection to create a processed nucleic acid template library, wherein the adaptor includes a first DNA sequence encoding a PAM sequence and at least a tracr mate.