Nucleic Acid Concentration Correction for Fragmented Sequence Amplification
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Solution Overview
Problem
Existing methods for measuring nucleic acid sequence concentrations in biological samples fail to accurately quantify fragmented nucleic acids, leading to underestimation due to incomplete amplification of sequences shorter than the target length.
Innovation Solution
A method is developed to correct the measured concentration of fragmented nucleic acids by using a correction coefficient based on the length distribution and parameters of the measuring method, allowing for the determination of non-fragmented nucleic acid concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR amplification is used to measure nucleic acid sequence concentrations, then amplification efficiency is improved for complete sequences, but measurement accuracy deteriorates for fragmented sequences shorter than the target length
Solution Approach 1:
The invention changes the parameter of sequence length by designing multiple primer pairs that target different length regions of the same nucleic acid sequence. This allows fragmented sequences of varying lengths to be amplified and detected, thereby improving measurement accuracy for degraded samples while maintaining amplification efficiency through optimized primer design and selection.
2Speed
If standard PCR conditions are applied, then amplification speed is maintained, but underestimation of fragmented nucleic acids occurs
Solution Approach 1:
The invention segments the amplification process by using multiple primer pairs with different annealing temperatures and binding sites. This segmentation allows parallel amplification of sequences of different lengths and compositions, improving quantification reliability for fragmented nucleic acids while maintaining overall amplification speed through optimized cycling conditions.
Solution Approach 2:
The invention applies partial action by using a subset of primer pairs in different PCR reactions rather than attempting to amplify all sequences simultaneously in one reaction. This approach allows optimization of each reaction for specific sequence types while maintaining overall amplification efficiency and improving quantification 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
The method provides a more accurate estimation of nucleic acid concentrations by accounting for incomplete amplification of fragmented sequences, improving the reliability of quantitative analysis.
Implementation Method 1
Amplification can be carried out by combining oligonucleotide primers with the sample and then subjecting the sample to amplification conditions compatible with nucleic acids quantification, such as polymerase chain reaction (PCR) conditions.
Data Source
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AI summary
The present invention relates to a method of determining the concentration of a detected sequence in non-fragmented nucleic acids by applying a correction coefficient to the concentration of said detected sequence measured in fragmented nucleic acids.