Unbiased and simultaneous amplification method for preparing a double-stranded DNA library from a sample of more than one type of nucleic acid
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Solution Overview
Problem
Conventional methods for preparing DNA libraries from samples with low amounts of mixed nucleic acids, such as DNA and RNA, often introduce bias and require complex, time-consuming procedures, failing to simultaneously amplify different types of nucleic acids effectively.
Innovation Solution
A method involving the use of carefully designed DNA strand generation primers with specific nucleotide sequences and a polymerase for one-pot synthesis, followed by amplification and fragmentation using methylation-specific restriction enzymes, to generate unbiased double-stranded DNA libraries from mixed nucleic acid samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional amplification methods are used on mixed nucleic acid samples, then amplification can be achieved, but bias is introduced between different types of nucleic acids
Solution Approach 1:
The method segments the amplification process into distinct phases: first-strand synthesis using oligo-dT primers for RNA and random primers for DNA, followed by second-strand synthesis. This segmentation allows each primer type to target specific nucleic acid forms, eliminating cross-reactivity bias and enabling unbiased simultaneous amplification of multiple nucleic acid types.
Solution Approach 2:
The patent employs a universal amplification system that can process multiple nucleic acid types (DNA, RNA, ssDNA, dsDNA) through a single protocol. The combination of oligo-dT primers and random primers creates a multi-functional primer pool that universally binds to different nucleic acid templates, achieving versatility without sacrificing amplification reliability.
2Quantity of substance
If complex procedures are used to amplify low amount of nucleic acids, then amplification sensitivity is improved, but time consumption and cost increase
Solution Approach 1:
The method merges multiple amplification targets into a single reaction pot, simultaneously amplifying DNA, RNA, and their analogues in one procedure. This consolidation eliminates the need for separate processing steps for different nucleic acid types, significantly reducing time consumption and operational costs while maintaining high sensitivity for low-input samples.
Solution Approach 2:
The patent optimizes reaction parameters including primer concentrations, polymerase selection, and thermal cycling conditions to achieve efficient amplification of low-abundance nucleic acids. By adjusting these parameters, the method achieves high sensitivity without requiring complex pre-enrichment or concentration steps, thereby reducing overall procedure time.
3Productivity
If random primers with few random nucleotides are used, then PCR efficiency is improved, but cross-hybridization among primers increases
Solution Approach 1:
The patent applies local quality by designing primers with differentiated random nucleotide regions. Each primer contains a unique combination of random nucleotides (e.g., 2-6 random bases) that provides sufficient binding variability to prevent cross-hybridization, while maintaining short overall primer length to preserve PCR efficiency. This localized differentiation in primer sequences eliminates harmful cross-reactivity without sacrificing productivity.
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 method enables efficient, unbiased amplification and library preparation from low amounts of mixed nucleic acids, reducing bias and the formation of non-specific amplicons, and facilitates subsequent sequencing by fragmenting amplicons into manageable sizes for barcoding and analysis.
Implementation Method 1
extending from the annealed first DNA strand generation primer including employing a DNA polymerase that enables a one-pot synthesis of the first DNA strand from either or both of DNA and/or RNA templates
Implementation Method 2
employing a DNA polymerase having strand displacement activity, wherein said second DNA strand generation primer includes: a random nucleotide sequence including a plurality of said random nucleotides at 3′-end
Implementation Method 3
fragmenting the amplicons into a plurality of double-stranded DNA fragments including reacting the amplicons with a methylation-specific restriction enzyme
Implementation Method 4
each of the first and second adaptor sequences has at least one nucleotide modified by methylation
Data Source
AI summary
The present invention relates to an unbiased and simultaneous amplification method for preparing a double-stranded DNA library from a sample of more than one type of nucleic acid. In particular, the present invention provides an unbiased and simultaneous amplification method for preparing a double-stranded DNA library from more than one type of nucleic acid in substantially low amount comparative to non-nucleic acid molecules in the sample within a relatively shorter turnaround time and substantially without any purification step between amplifications and between cDNA preparation and amplification as compared to conventional methods of preparing DNA library.