Random Amplification of Low-Input Nucleic Acids for NGS
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Solution Overview
Problem
Current methods for nucleic acid amplification, such as next-generation sequencing, require significant amounts of DNA or RNA, often exceeding what is present in clinical samples, making it difficult to directly sequence viral or microbial nucleic acids from samples like blood, respiratory secretions, and cerebrospinal fluid without culture techniques.
Innovation Solution
A one-step or two-step method for random amplification of low-input nucleic acids using a combination of reverse transcriptase, thermostable DNA polymerase lacking 5′ to 3′ proofreading exonuclease activity, and hot-start PCR polymerase, with specific oligonucleotides and thermocycling steps, allowing for amplification of as little as 0.005 pg of input nucleic acid in a single tube or two tubes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If next-generation sequencing is used to detect viral or microbial nucleic acids, then sequencing capability is provided, but the method requires 0.1 ng to 10 μg of DNA or cDNA which exceeds the amount present in clinical samples (less than 0.001 ng)
Solution Approach 1:
The patent applies preliminary action by performing random amplification of nucleic acids before sequencing. The method uses a multi-enzyme system (reverse transcriptase, thermostable DNA polymerase lacking 5' to 3' proofreading exonuclease activity, and hot-start PCR polymerase) with specific oligonucleotides to amplify extremely low quantities of input nucleic acid (less than 1 pg) to sufficient levels for NGS library preparation, thereby resolving the contradiction between low sample quantity and sequencing requirements
2Adaptability or versatility
If microorganism-specific primers are used for amplification, then specific pathogen detection is achieved, but the method cannot detect unknown microorganisms without prior sequence knowledge
Solution Approach 1:
The patent applies universality by using random amplification with a multi-enzyme system and specific oligonucleotide structures (including primer pairs with defined sequences and lengths) that can amplify any nucleic acid template without requiring pathogen-specific primers. This universal approach enables detection of both known and unknown microorganisms while maintaining reliability through the specificity of the enzymatic reactions and thermocycling conditions
3Quantity of substance
If complex sample preparation protocols are used to amplify low-input nucleic acids, then amplification sensitivity is improved, but hands-on time and procedural complexity increase
Solution Approach 1:
The patent applies merging by combining multiple enzymatic activities (reverse transcription, DNA synthesis, and PCR amplification) into a single integrated reaction system. The method uses a specific combination of enzymes (reverse transcriptase, thermostable DNA polymerase lacking 5' to 3' proofreading exonuclease activity, and hot-start PCR polymerase) with defined oligonucleotides that can be mixed in a single container and processed through standardized thermocycling, thereby achieving high amplification sensitivity while minimizing hands-on time and procedural complexity
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 the amplification of extremely low quantities of nucleic acids to sufficient levels for next-generation sequencing, eliminating the need for microorganism-specific primers and allowing for the amplification of unknown microorganisms without prior sequence knowledge, with minimal hands-on time and requiring only a standard PCR machine.
Implementation Method 1
a first synthesis step to permit the reverse transcriptase to generate first strand cDNA
Implementation Method 2
a second synthesis step to permit the thermostable DNA polymerase to generate second strand cDNA
Implementation Method 3
PCR using the first oligonucleotide as primer and the second strand cDNA as template
Implementation Method 4
a denaturation step to denature the first strand cDNA from the input nucleic acid and inactivate the reverse transcriptase
Data Source
AI summary
Methods for the rapid amplification of extremely low quantity nucleic acids in a sample are provided. The disclosed methods are capable of amplifying less than 1 pg of DNA and/or RNA from a biological sample using a single tube and one-step or two-step preparation.