Lysis Buffer for Nucleic Acid Extraction from Solid Samples
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Solution Overview
Problem
The extraction of nucleic acid from solid biological materials like calcified bone and tooth, as well as from biological samples adherent or embedded in adhesive and denim materials, poses challenges due to potential PCR inhibitors and requires efficient methods for DNA preservation and removal of inhibitors to facilitate downstream applications such as genotyping and identification.
Innovation Solution
A lysis solution comprising a detergent, a chelating agent, a reducing agent, and an enzyme is used to effectively extract nucleic acids from solid biological samples, adhesive materials, and denim substrates, which can include N-lauroyl sarcosine, ethylene glycol tetraacetic acid, tris(2-carboxyethyl)phosphine, and proteinase K, allowing for high-yield DNA recovery and removal of PCR inhibitors, and is automatable using standard liquid handling systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional extraction methods are used for solid biological materials, then the extraction process is simple, but the yield of nucleic acid is low and PCR inhibitors are present
Solution Approach 1:
The patent modifies the chemical parameters of the lysis buffer by incorporating specific detergents (SDS, CHAPS), chelating agents (EDTA, EGTA), reducing agents (DTT, TCEP), and enzymes (proteinase K, benzonase) to optimize nucleic acid release from solid matrices while maintaining compatibility with downstream PCR applications
Solution Approach 2:
The lysis buffer comprises a composite formulation combining multiple functional components (detergents, chelating agents, reducing agents, enzymes, and buffering agents) that work synergistically to achieve high-yield nucleic acid extraction from diverse solid samples including bone, tooth, adhesive materials, and denim
2Reliability
If standard lysis buffers are used, then the buffer composition is simple, but DNA integrity is not preserved and inhibitors are not removed
Solution Approach 1:
The patent optimizes buffer parameters including pH (maintained at 8.0-8.5 using Tris-HCl), ionic strength (150-500 mM NaCl), and component concentrations to preserve DNA integrity while effectively removing inhibitors through chelation and reduction reactions
Solution Approach 2:
The lysis buffer acts as an intermediary solution that facilitates the transition from intact solid biological samples to purified nucleic acids suitable for PCR, mediating the breakdown of cellular structures while protecting DNA from degradation
3Productivity
If manual extraction procedures are used, then the equipment requirement is low, but the processing time is long and labor-intensive
Solution Approach 1:
The lysis buffer formulation enables self-contained sample processing where the buffer components automatically perform lysis, inhibitor removal, and DNA protection functions without requiring complex equipment or multiple manual intervention steps
Solution Approach 2:
The lysis buffer serves multiple functions simultaneously: cell lysis via detergents, protein digestion via enzymes, metal ion chelation via EDTA/EGTA, reduction of disulfide bonds via DTT/TCEP, and DNA stabilization, making it universally applicable to diverse solid sample types
4Adaptability or versatility
If conventional buffers are used for adhesive and denim samples, then the buffer is compatible with standard protocols, but PCR inhibitors are extracted with the nucleic acid
Solution Approach 1:
The lysis buffer components act as intermediaries that selectively bind or neutralize PCR inhibitors (such as humic acids, tannins, and adhesive residues) while allowing nucleic acids to remain in solution for downstream processing
Solution Approach 2:
The patent converts potentially harmful interactions between conventional buffers and inhibitors into beneficial effects by using chelating agents to bind metal-dependent inhibitors and reducing agents to modify inhibitor structures, thereby eliminating their harmful effects while maintaining buffer compatibility
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 solution enables efficient extraction and purification of nucleic acids with improved yield and reduced inhibitor presence, facilitating sensitive DNA detection and genotyping, even from challenging samples like bone, tooth, and denim materials, and is suitable for forensic and research applications.
Implementation Method 1
The detergent can be an anionic detergent, a cationic detergent, a nonionic detergent, or a combination thereof
Implementation Method 2
The chelating agent can be one or more of ethylene glycol tetraacetic acid (EGTA) and ethylene diamine tetraacetic acid (EDTA), citric acid and combinations thereof
Implementation Method 3
The reducing agent can be one or more of tris(2-carboxyethyl)phosphine (TCEP), dithioerythritol (DTE), and dithiothreitol (DTT)
Implementation Method 4
The enzyme can be one or more of caspase, chymotrypsin, pepsin, proteinase K, thrombin, Staphylococcus V8 protease, pronase, papain, Bacillus sp. E1A protease, and trypsin
Data Source
AI summary
The present teachings relate to the extraction of nucleic acid from solid materials. Provided are useful compositions, methods, and kits for obtaining nucleic acids from a solid biological sample or an adhesive material having a biological material adherent or embedded within the adhesive substrate. The extracted nucleic acid can be used in downstream applications such as genotyping, detection, quantification, and identification of the source of the biological material.