Multiplexed PCR Normalization for DNA Library Screening
Solution Overview
Problem
Current multiplexed target-binding candidate screening analysis systems face difficulties in simultaneously selecting many nucleotide-containing peptide libraries for binding to a desired target due to sample-to-sample variations and data complexity.
Innovation Solution
A method and system for normalizing polymerase chain reaction (PCR) amplification processes by sorting DNA-containing compositions into bins based on common PCR cycle counts, allowing simultaneous PCR performance across libraries to achieve normalization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional PCR amplification is performed on multiple samples simultaneously, then throughput is improved, but amplification bias and inhibition occur due to varying DNA concentrations and sample qualities
Solution Approach 1:
The patent divides the PCR amplification process into separate reactions for different sample groups. Samples are partitioned into multiple wells, each containing a subset of samples that are amplified simultaneously in separate reactions. This segmentation allows each reaction to proceed with controlled conditions, preventing inhibition and bias that would occur in a single mixed reaction, while still maintaining high throughput through parallel processing of multiple groups.
Solution Approach 2:
The patent employs digital normalization where cycle threshold (Ct) values are transformed into digital quantities through mathematical operations. The system calculates normalized copy numbers by adjusting Ct values based on reference samples and mathematical transformations (such as 2^(-Ct) calculations). This parameter transformation enables quantitative comparison across samples with different starting concentrations and qualities, maintaining amplification accuracy while processing multiple samples simultaneously.
2Adaptability or versatility
If DNA concentration varies between samples, then sample diversity is improved, but amplification inhibition and bias increase
Solution Approach 1:
The patent incorporates reference samples with known DNA concentrations into each PCR reaction group. These reference samples provide a feedback mechanism that allows the system to normalize the amplification results. By comparing the Ct values of test samples against the reference samples in the same reaction, the system can calculate normalization factors that compensate for variations in DNA concentration and inhibition effects, maintaining consistent amplification across diverse samples.
Solution Approach 2:
The patent uses reference samples as intermediaries to bridge the differences between samples with varying DNA concentrations. The reference samples act as mediators that standardize the amplification process by providing a common baseline for comparison. Through the calculation of normalized copy numbers using reference sample data, the system eliminates the direct impact of concentration variations and inhibition effects, enabling reliable analysis of diverse samples.
3Productivity
If multiple samples are processed in parallel, then productivity is improved, but data normalization and comparison become more complex
Solution Approach 1:
The patent creates a universal normalization method that can be applied across all sample groups regardless of their specific characteristics. The same mathematical approach (using reference samples and digital transformations) is used for all reactions, simplifying the normalization process. This universal approach allows the system to handle multiple samples in parallel without increasing operational complexity, as the same standardized procedure applies to each group.
Solution Approach 2:
The patent transforms amplification data from continuous Ct values into discrete digital normalized copy numbers through mathematical operations. This parameter transformation simplifies data comparison and normalization across multiple parallel samples. By converting all measurements to a common digital scale based on reference samples, the system eliminates the need for complex individual normalization calculations for each sample, reducing computational complexity while maintaining high processing throughput.
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 reduces variation between samples, enabling reliable and reproducible multiplexed screening of nucleotide-tagged peptide libraries for target-binding activity, facilitating robust tracking of selection progress across multiple rounds.
Implementation Method 1
a method and system for normalizing a polymerase chain reaction (PCR) amplification process for a plurality of samples containing DNA
Data Source
Figure 1
Figure 2
Figure 3A
AI summary
A method and system for normalizing a polymerase chain reaction (PCR) amplification process for a plurality of samples containing DNA. Cycle data for performing the PCR amplification process is identified for the plurality of samples. The cycle data includes a corresponding cycle count for each sample of the plurality of samples. The plurality of samples is sorted into a plurality of bins based on the cycle data such that cycle count variation between bins of the plurality of bins is reduced. A bin cycle count is assigned to each bin of the plurality of bins. The bin cycle count is unique to each bin of the plurality of bins. Identification information is generated for the bins. An output for performing a PCR amplification of the plurality of samples is generated using the bins and the bin cycle count for each bin of the plurality of bins.