Isothermal Nucleic Acid Amplification via Nicking Enzyme Cleavage
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Solution Overview
Problem
Current isothermal strand displacement amplification methods require denaturation of double-stranded DNA to single strands, limiting their efficiency and specificity, especially when natural nicking sites are not present in the target region of interest.
Innovation Solution
The method involves using specifically designed primers with a non-complementary tail containing a nicking enzyme recognition sequence, paired with a polymerase enzyme and a nicking enzyme, to facilitate isothermal amplification at temperatures between 45°C and 55°C without prior denaturation of the double-stranded genomic nucleic acid.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If heat denaturation at 95°C is used to separate dsDNA into single strands, then primer binding is improved, but amplification efficiency and specificity are reduced due to non-specific binding and loss of target integrity
Solution Approach 1:
The patent changes the temperature parameter from high (95°C) to low (45-55°C) by using nicking enzymes that recognize specific sequences on double-stranded DNA, allowing strand displacement without heat denaturation. This maintains target integrity while enabling specific amplification.
Solution Approach 2:
The patent replaces the thermal mechanical system (heat denaturation) with a biochemical system (nicking enzyme recognition and cleavage). The nicking enzyme specifically binds to recognition sequences and cleaves one strand, allowing primer extension without high temperature.
2Productivity
If pre-denaturation is required to generate ssDNA targets, then primer extension can proceed, but the method becomes less efficient and requires additional time and energy
Solution Approach 1:
The patent incorporates the nicking enzyme recognition sequence directly into the primer design, so that when the primer binds to the target, it simultaneously presents the recognition sequence to the nicking enzyme. This eliminates the need for separate denaturation steps and enables continuous amplification.
Solution Approach 2:
The patent establishes a continuous amplification cycle where nicking enzyme cleavage, primer extension, and new nicking site generation occur sequentially without interruption. The isothermal conditions allow the reaction to proceed continuously at 45-55°C without thermal cycling.
3Reliability
If natural nicking sites are not present in the target region, then specific amplification cannot occur, but the method is limited by enzyme availability
Solution Approach 1:
The patent performs preliminary action by incorporating the nicking enzyme recognition sequence into the primer design itself. This ensures that every amplification event creates a new nicking site, eliminating the dependency on pre-existing natural nicking sites in the target DNA.
Solution Approach 2:
The patent makes the primer multi-functional by combining three functions in one oligonucleotide: (1) target binding through complementarity, (2) providing the nicking enzyme recognition sequence, and (3) serving as the template for extension. This universal design works with any target sequence.
4Stability of the object's composition
If double-stranded DNA is used as target, then sample integrity is maintained, but primer binding efficiency is reduced without denaturation
Solution Approach 1:
The patent introduces the nicking enzyme as an intermediary that binds to the double-stranded DNA at the recognition sequence and cleaves one strand. This creates a single-stranded region that allows primer binding while the rest of the DNA remains double-stranded and intact.
Solution Approach 2:
The patent applies local quality change by maintaining double-stranded structure throughout most of the DNA molecule while creating a localized single-stranded region at the nicking site where primer binding occurs. This preserves overall DNA integrity while enabling specific binding.
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 enables efficient and specific amplification of target nucleic acids without denaturation, allowing for the detection of clinical relevant target levels in biological samples with high sensitivity and specificity, and can be combined with other amplification methods for enhanced performance.
Implementation Method 1
a nicking enzyme specific for the nicking enzyme recognition sequence
Implementation Method 2
a polymerase enzyme having strand displacement activity
Implementation Method 3
the forward primer has the formula: A-B, wherein B comprises a portion of the forward primer that is complementary to the target nucleic acid sequence
Data Source
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AI summary
Methods, primers and probes are provided for the isothermal amplification and detection, without denaturation, of double stranded nucleic acid targets for polymerase strand displacement amplification ("iSDA"), The methods and compositions disclosed are highly specific for nucleic acid targets with high sensitivity, specificity and speed that allow detection of clinical relevant target levels. The methods and compositions can easily be used to amplify or detect nucleic acid targets in biological samples.