Single Strand Annealing Proteins Enhance Nucleic Acid Ligation Efficiency
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Solution Overview
Problem
Current methods for nucleic acid ligation and hybridization suffer from low efficiency and sensitivity, particularly in biological sample analysis, leading to challenges in accurately detecting and analyzing target nucleic acid sequences.
Innovation Solution
The use of single strand annealing proteins (SSAPs) such as DdrB or RecT to bind and facilitate the hybridization and ligation of nucleic acid sequences, significantly increasing ligation efficiency and fidelity by promoting the annealing of nucleic acid strands to a third nucleic acid sequence acting as a template.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional nucleic acid ligation methods are used, then the process is simple, but ligation efficiency and sensitivity are low
Solution Approach 1:
The patent introduces single strand annealing proteins (SSAPs) as intermediary molecules that mediate between the nucleic acid sequences and the ligation process. These proteins bind to single-stranded nucleic acids and facilitate their annealing to a template strand, thereby enhancing ligation efficiency without requiring complex equipment or procedures
Solution Approach 2:
The patent changes the chemical and physical parameters of the ligation system by introducing proteins that alter the binding affinity and annealing kinetics of nucleic acid strands. This modifies the reaction conditions to favor more efficient and specific ligation, improving both efficiency and sensitivity
2Measurement precision
If conventional hybridization methods are used, then the procedure is straightforward, but hybridization efficiency and accuracy are insufficient
Solution Approach 1:
Single strand annealing proteins serve as mediators that enhance the specificity and efficiency of hybridization. These proteins bind to single-stranded nucleic acids and facilitate their accurate pairing with complementary template strands, improving both the precision and speed of hybridization reactions
3Reliability
If ligation is performed without SSAPs, then the reaction conditions are simple, but sensitivity for biological sample analysis is low
Solution Approach 1:
The patent employs single strand annealing proteins as intermediary agents that significantly enhance the sensitivity of nucleic acid detection in biological samples. These proteins improve the efficiency of ligation and hybridization reactions, enabling more reliable detection of target sequences even at low concentrations
Solution Approach 2:
The single strand annealing proteins perform preliminary binding and annealing actions before the actual ligation step. This preliminary action ensures that nucleic acid strands are properly positioned and stabilized, increasing the likelihood of successful ligation and thereby improving overall detection sensitivity
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 approach enhances ligation efficiency by up to 100% and hybridization efficiency, leading to improved sensitivity and accuracy in nucleic acid analysis, especially in biological samples, and enables more efficient generation of full-length barcoded oligonucleotides in multiple rounds of ligation.
Implementation Method 1
the first nucleic acid sequence hybridizes to a first hybridization region of a third nucleic acid sequence and the second nucleic acid sequence hybridizes to a second hybridization region of the third nucleic acid sequence
Implementation Method 2
single strand annealing protein (SSAP)...promote the annealing of nucleic acid strands to a third nucleic acid sequence acting as a template
Data Source
AI summary
The present disclosure relates in some aspects to methods and compositions for analyzing a target nucleic acid in a biological sample. In some aspects, the methods disclosed herein promote hybridization of polynucleotides to target nucleic acids and/or splints for enhanced ligation efficiency and/or enhanced hybridization efficiency in situ in a biological sample. In some aspects, the presence, amount, and/or identity of a target nucleic acid is analyzed in situ. Also provided are compositions and kits for use in accordance with the methods.