Selective DNA Amplification Using dSaCas9 Blocking in MDA
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Solution Overview
Problem
Existing nucleic acid amplification methods, such as PCR and MDA, struggle to selectively amplify target DNA sequences like pathogen DNA over non-target human DNA, especially in complex samples, due to the lack of effective blocking agents that can withstand the displacement by highly progressive polymerases like Φ29DNA polymerase, and the salt incompatibility issues with Cas proteins.
Innovation Solution
Employing a catalytically dead Staphylococcus aureus Cas9 (dSaCas9) complexed with guide RNAs to selectively bind to human DNA sequences, followed by strand-displacing DNA polymerase under different salt conditions, allowing dSaCas9 to initially bind strongly and then maintain blocking while Φ29DNA polymerase amplifies target DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If a blocking oligonucleotide primer is used to suppress amplification of non-target sequences, then amplification specificity is improved, but the blocking primer is displaced by highly progressive polymerases like Φ29DNA polymerase during MDA
Solution Approach 1:
The patent changes the chemical parameters of the blocking agent by using a DNA molecule with a modified backbone (such as a phosphorothioate linkage or other chemically modified nucleic acid) that confers resistance to strand extension by Φ29DNA polymerase. This modified blocking DNA remains stable during the MDA process and effectively prevents amplification of non-target sequences without being displaced by the highly progressive polymerase.
Solution Approach 2:
The invention employs a composite blocking system where a chemically modified DNA molecule (with resistant backbone) is combined with the MDA reaction components. This composite approach integrates the blocking function with the polymerase resistance, creating a stable blocking agent that maintains its position throughout the amplification process.
2Manufacturing precision
If Cas proteins are used to block amplification, then selectivity is improved, but salt incompatibility issues arise with Φ29DNA polymerase
Solution Approach 1:
The patent extracts the salt-sensitive Cas protein component and replaces it with a salt-tolerant alternative. The blocking function is achieved using a chemically modified DNA molecule that does not depend on Cas proteins, thereby eliminating the salt incompatibility issue while maintaining amplification selectivity.
Solution Approach 2:
The invention uses a simple, chemically modified DNA molecule as the blocking agent instead of complex Cas proteins. This simplified approach employs a disposable-like single-use modified DNA molecule that provides the necessary blocking function without the complexity and salt sensitivity of protein-based systems.
3Length of moving object
If random hexamer primers are used for amplification, then amplification of long sequences is achieved, but non-target sequences are amplified without selectivity
Solution Approach 1:
The patent segments the amplification process into two functional components: (1) random hexamer primers that provide non-specific binding and enable amplification of long sequences, and (2) a chemically modified blocking DNA that provides selectivity by preventing amplification of non-target sequences. This segmentation allows each component to perform its specific function without interfering with the other.
Solution Approach 2:
The chemically modified blocking DNA acts as an intermediary element that mediates between the random hexamer primers and the target sequences. It selectively binds to non-target sequences and prevents their amplification, while allowing the random hexamer primers to continue amplifying target sequences of interest.
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
Achieves selective and efficient amplification of pathogen DNA over human DNA, producing amplified and enriched DNA suitable for whole genome sequencing by ensuring dSaCas9 remains bound during Φ29DNA polymerase activity, overcoming salt incompatibility and displacement challenges.
Implementation Method 1
a catalytically dead Staphylococcus aureus Cas9 (dSaCas9) complexed with guide RNAs having selective binding affinity for DNA sequences preferentially present in the first population of DNA compared to the second population of DNA
Implementation Method 2
Suitable amplification techniques for long run amplifications typically use highly progressive strand displacement nucleic acid polymerases such as phi (Φ) DNA polymerase which can produce very large DNA amplification with a low error rate in a process known as multiple displacement amplification (MDA)
Implementation Method 3
guide RNAs having selective binding affinity for DNA sequences preferentially present in the first population of DNA
Data Source
AI summary
A method of selective DNA amplification of a DNA mixture comprising a first population of DNA and a second population of DNA, wherein:—a) a catalytically dead Staphylococcus aureus Cas9 complex (dSaCas9) comprising a dSaCas9 protein or derivative thereof complexed with one or more guide RNAs having selective binding affinity for DNA sequences preferentially present in the first population of DNA compared to the second population of DNA is contacted with the DNA mixture under a first reaction condition, said first reaction condition being suitable for binding of the dSaCas9 complex to DNA sequences for which it has a binding affinity, and thenb) a strand-displacing DNA polymerase is contacted with the DNA mixture under a second reaction condition, said second reaction condition being suitable for amplification being suitable for amplification activity of the strand-displacing DNA polymerase; and related kits and uses.