Lysis Mixture for In Situ Nucleic Acid Analysis
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current procedures for real-time PCR and qRT-PCR on cell lysates are not suitable for high-throughput analysis due to temperature control challenges and the presence of inhibitory components that affect reverse transcriptase and DNA polymerase function.
Innovation Solution
A process involving a lysis mixture with protease and deoxyribonuclease activities, along with a surfactant like TRITON X-114, TRITON X-100, or NONIDET P-40, that is free of cation chelators, combined with a stop mixture containing a cation chelator to inactivate enzymes, allowing for efficient sample preparation at ambient temperatures compatible with reverse transcription and PCR reactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current procedures for RT-PCR on cell lysates are used, then nucleic acid detection can be performed, but temperature control across the entire plate becomes challenging and throughput is limited
Solution Approach 1:
The procedure is divided into separate temperature-controlled steps: an initial lysis/incubation step at elevated temperature (50-65°C) followed by a second step at lower temperature (4-25°C) for reverse transcription and PCR. This segmentation allows each step to be optimized independently and facilitates high-throughput processing.
Solution Approach 2:
Cell lysis and nucleic acid release are performed in advance during the first temperature step before the actual RT-PCR reaction begins. This preliminary action prepares the sample in advance, allowing the subsequent amplification steps to proceed efficiently without requiring continuous temperature adjustments.
2Reliability
If current RT-PCR procedures are used, then nucleic acid detection is achieved, but components in the procedure are inhibitory for reverse transcriptase and DNA polymerase function
Solution Approach 1:
The procedure separates the lysis step from the RT-PCR reaction step. During lysis, cellular components are released but not yet mixed with the enzymatic reaction mixture. This extraction approach allows harmful components to be diluted or removed before the enzymatic reactions begin, improving enzyme activity and reliability.
Solution Approach 2:
A buffer solution is introduced as an intermediary medium that facilitates the transition from lysis to RT-PCR. This buffer is optimized to neutralize inhibitory components while maintaining conditions favorable for reverse transcriptase and DNA polymerase activity, thereby improving enzymatic reliability.
3Productivity
If traditional RNA purification methods are used, then high purity RNA is obtained, but the process is time-consuming and not suitable for high throughput
Solution Approach 1:
The procedure merges cell lysis, nucleic acid release, and RT-PCR setup into a single integrated workflow without requiring separate purification steps. This combination maintains sufficient RNA purity for detection while dramatically reducing processing time and enabling high-throughput analysis.
Solution Approach 2:
The lysis buffer and reaction conditions are designed to work directly with crude cell lysates without requiring additional purification steps. The system is self-sufficient, using the lysate as-is while the enzymatic reactions and detection methods are optimized to tolerate residual contaminants, thereby eliminating time-consuming purification steps.
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
Enables fast, efficient, and ambient temperature production of RT-PCR ready lysates, facilitating high-throughput analysis and maintaining enzymatic activity, with results comparable to traditional RNA purification methods.
Implementation Method 1
The lysis mixture comprises a polypeptide having protease activity
Implementation Method 2
a polypeptide having deoxyribonuclease activity
Implementation Method 3
a surfactant that substantially lacks fluorescence between 300 nm and 750 nm when in use for in situ analysis of RNA or a surrogate thereof
Implementation Method 4
The stop mixture comprises a cation chelator effective to inactivate the polypeptide having deoxyribonuclease activity
Implementation Method 5
an inhibitor of the polypeptide having protease activity
Implementation Method 6
The resultant stopped mixture is compatible with in situ reverse transcriptase and DNA polymerase reactions
Implementation Method 7
DNA polymerase reactions
Data Source
AI summary
Sample preparation processes for in situ RNA or DNA analysis, methods and compositions therefor are provided. Processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or extraction. The preparation process can be carried out at room temperature in as little as seven minutes and is amenable to high throughput processing using manual or robotic platforms.