Direct Nucleic Acid Amplification from Crude Samples
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Solution Overview
Problem
Current PCR amplification methods require complex purification steps and large sample volumes, leading to significant nucleic acid loss and analysis bias, especially for minute amounts of cell-free nucleic acids in body fluids and forensic samples.
Innovation Solution
A method for direct adapter ligation and PCR amplification from crude samples without pre-purification, involving dilution, protein denaturation, end modification, and adapter ligation using double-stranded homogeneous adapters, allowing amplification of nucleic acids in a single drop of sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If pre-purification of nucleic acids is performed, then the quality of nucleic acid analysis is improved, but the quantity of nucleic acid molecules is significantly reduced (loss >20%)
Solution Approach 1:
The patent extracts and removes inhibitory substances (proteins, salts, metabolites) from the crude sample through protein denaturation and precipitation steps, separating them from the nucleic acids to enable direct amplification without requiring complete purification, thus avoiding the >20% loss associated with conventional purification
Solution Approach 2:
The patent performs preliminary protein denaturation and inhibitor removal steps before adapter ligation and PCR amplification, preparing the sample in advance to eliminate interfering substances that would otherwise inhibit the amplification process, thereby enabling direct amplification from crude samples
2Measurement precision
If pre-isolation of nucleic acids is performed, then the purity of nucleic acid molecules is improved, but the quantity of nucleic acid molecules is significantly reduced (loss >20%)
Solution Approach 1:
The patent extracts and removes inhibitory substances (proteins, salts, metabolites) from the crude sample through protein denaturation and precipitation steps, separating them from the nucleic acids to enable direct amplification without requiring complete purification, thus avoiding the >20% loss associated with conventional purification
Solution Approach 2:
The patent uses protein denaturation agents and precipitation reagents as intermediaries to selectively remove inhibitory substances from the sample, allowing the nucleic acids to remain in solution and be directly amplified without undergoing loss-prone purification steps
3Measurement precision
If conventional PCR amplification is performed on minute amounts of nucleic acids, then the sensitivity is improved, but the complexity of the process increases due to required purification steps
Solution Approach 1:
The patent merges the sample preparation steps (protein denaturation, inhibitor removal) with the PCR amplification process, combining what were previously separate sequential operations into an integrated workflow that enables direct amplification from crude samples without requiring separate purification steps
Solution Approach 2:
The patent enables the crude sample to serve directly as the template for PCR amplification after simple protein denaturation, eliminating the need for complex external purification procedures and allowing the sample matrix to be processed in-place during the amplification workflow
4Reliability
If pre-isolation of nucleic acids is performed, then the reliability of nucleic acid analysis is improved, but the time required for analysis increases
Solution Approach 1:
The patent extracts and removes inhibitory substances (proteins, salts, metabolites) from the crude sample through protein denaturation and precipitation steps, separating them from the nucleic acids to enable direct amplification without requiring complete purification, thus avoiding the >20% loss associated with conventional purification
Solution Approach 2:
The patent skips the time-consuming conventional purification steps (spin columns, magnetic beads, multiple washes) by implementing a streamlined approach where protein denaturation and inhibitor removal are achieved through simplified precipitation and filtration, dramatically reducing the time required before amplification
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 simplifies nucleic acid amplification, reduces sample requirements, and enhances sensitivity and accuracy, overcoming the limitations of conventional PCR techniques for analyzing minute nucleic acid amounts in body fluids and other samples.
Implementation Method 1
adding the mixed solution into a test tube, heating the mixed solution in the test tube to denature proteins
Implementation Method 2
performing a ligation reaction between the processed nucleic acid fragments and double-stranded adapters
Implementation Method 3
performing the adapter-dependent PCR on the ligated nucleic acid fragments
Data Source
AI summary
A method for amplifying free nucleic acids (NAs) directly from one drop unpurified crude sample. The sample comprises nucleic acid fragments. The method includes (a) mixing an unpurified sample with a buffer to form an uniformly mixed solution; (b) adding the mixed solution to a tube then heating the tube to denature the proteins in the mixed solution, and cooling to room temperature; (c) subjecting he nucleic acid fragments in the mixture to a processing reaction required for adaptor-dependent PCR; (d) performing a ligation reaction between the processed nucleic acid fragments and adapters, forming nucleic acid fragments ligated with adapter in both ends, wherein the adapter is a complementary double-stranded nucleic acid (dsNA) fragment, one of which is an oligonucleotide with 5′-phosphate and the other is an oligonucleotide with thymine (T) or uracil (U); and (e) performing the adapter-dependent PCR to the nucleic acid fragments in the mixed solution.