Multiple Ligase Compositions for Complete DNA Library Ligation
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Solution Overview
Problem
Existing DNA ligation technologies, particularly in library preparation for next-generation sequencing, face inefficiencies in ligation reactions, especially when joining double-stranded oligonucleotide adaptors to DNA fragments, leading to incomplete ligation and potential bias in sequencing results.
Innovation Solution
Employing a combination of adenylation-deficient ATP-dependent ligases and other ATP- or NAD-dependent ligases to separately ligate pre-adenylated and non-adenylated DNA strands, bypassing rate-limiting steps and optimizing the ligation process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a single DNA ligase is used to ligate both strands in traditional ligation reactions, then the reaction procedure is simple, but ligation efficiency and completeness are insufficient
Solution Approach 1:
The invention divides the ligation process into two separate reactions: one for ligating the first strand using an adenylation-deficient ligase, and another for ligating the second strand using a different ligase. This segmentation allows each ligase to be optimized for its specific substrate and mechanism, thereby improving overall ligation efficiency and completeness while maintaining manageable procedural complexity through standardized reaction protocols.
2Speed
If pre-adenylated DNA sequences are used to bypass rate-limiting steps, then ligation speed increases, but the complexity of reaction setup increases
Solution Approach 1:
The invention employs pre-adenylated DNA sequences as substrates, where the adenylation step has been performed in advance before the ligation reaction. This preliminary action bypasses the rate-limiting adenylation step during the actual ligation process, significantly increasing ligation speed. The pre-adenylated substrates are prepared separately and then used in the ligation reaction, which maintains relatively simple reaction setup while achieving rapid ligation.
3Reliability
If multiple ligases are used to ligate separate strands, then ligation completeness improves, but the number of reagents and steps increases
Solution Approach 1:
The invention uses different ligases with specialized functions: an adenylation-deficient ligase for ligating the first strand (which does not require adenylation) and a different ligase for ligating the second strand. Each ligase is selected for its specific capability and optimal performance with its designated substrate. This multi-functionality approach ensures complete ligation of both strands while managing reagent complexity through careful selection of ligases with non-overlapping or complementary specificities.
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
Enhances ligation efficiency and completeness, reducing the number of PCR cycles required and minimizing bias in sequencing data by using a mixture of ligases to catalyze separate strand sealing, thereby improving the library preparation workflow.
Implementation Method 1
DNA ligases are divalent metal ion dependent enzymes that utilize ATP or NAD+ to catalyze phosphodiester bond formation between adjacent polynucleotide termini possessing a 3'-hydroxyl and a 5'-phosphate
Implementation Method 2
In the case of ATP-dependent DNA ligases, the first step (step 1) involves the attack on the α-phosphate of ATP by ligase, which results in release of pyrophosphate and formation of a ligase-AMP intermediate
Implementation Method 3
DNA ligases are divalent metal ion dependent enzymes that utilize ATP or NAD+ to catalyze phosphodiester bond formation between adjacent polynucleotide termini
Implementation Method 4
In the case of ATP-dependent DNA ligases, the first step (step 1) involves the attack on the α-phosphate of ATP by ligase
Data Source
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AI summary
Provided herein are compositions, systems, and methods using multiple ligases, wherein at least one of the ligases is an adenylation-deficient ATP-dependent ligase or an un-adenylated ATP-dependent ligase (e.g., present in an ATP free mixture). In certain embodiments, multiple ligases are used to ligate a pre-adenylated double stranded sequence to a non-adenylated double stranded sequence (e.g., the adenylation-deficient ATP-dependent ligase or un-adenylated ATP-dependent ligase ligates the first strand, and a second ligase ligates the second strand). In other embodiments, provided herein are mutant T4 ligases (e.g., K159S mutant or K159C mutant).