Restriction Enzyme Digestion for DOP-PCR Primer Removal in DNA Sequencing
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Solution Overview
Problem
Current DNA amplification methods, such as DOP-PCR, introduce mutations and add sequences to the ends of amplified fragments, making them unsuitable for next-generation sequencing due to the inclusion of fixed primer sequences and non-perfect annealing of degenerate oligo primers, which limits the efficiency and accuracy of sequence reads.
Innovation Solution
A method involving amplifying nucleic acid fragments with a primer containing a target primer sequence and a recognition site for a restriction enzyme, followed by digestion with the restriction enzyme to remove the primer sequence, thereby exposing the target sequence for sequencing, using enzymes like BpmI that cleave outside the recognition site.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If DOP-PCR is used to amplify trace amount of DNA, then amplification efficiency is improved, but mutations and sequence additions are introduced at the ends of amplified fragments
Solution Approach 1:
The invention extracts and removes the harmful primer sequences from the ends of amplified DNA fragments using restriction enzyme digestion. The restriction enzyme specifically recognizes and cleaves the primer sequences that were added during DOP-PCR amplification, thereby eliminating the source of mutations and sequence errors while preserving the integrity of the amplified target DNA.
Solution Approach 2:
The invention incorporates a recognition site for restriction enzyme into the primer sequence before amplification begins. This preliminary design allows the restriction enzyme to subsequently remove the primer sequences after amplification, preventing the propagation of primer-induced errors into the final sequencing library.
2Device complexity
If fixed 5′ primer sequence is used for amplification, then amplification process is simplified, but primer sequences are present at both ends of every amplification product
Solution Approach 1:
The invention removes the fixed primer sequences from the amplified products through restriction enzyme digestion. The restriction enzyme specifically targets and cleaves the primer sequences that were incorporated during amplification, thereby eliminating them from the final product while maintaining the simplicity of the amplification process itself.
Solution Approach 2:
The invention uses a restriction enzyme as an intermediary to remove the primer sequences. The restriction enzyme acts as a mediator that specifically recognizes the primer sequences and cleaves them, providing a clean separation between the desired amplified product and the unwanted primer sequences.
3Quantity of substance
If degenerate oligo primers are used for library synthesis, then trace DNA can be amplified, but non-perfect annealing causes mutations and sequence additions
Solution Approach 1:
The invention extracts and removes the sequences added by degenerate oligo primers through restriction enzyme digestion. By cleaving the primer sequences that were added during library synthesis, the method eliminates the mutations and sequence additions caused by non-perfect annealing, while preserving the amplified target sequences.
Solution Approach 2:
The invention converts the harmful effect of primer sequence additions into a beneficial feature by designing the primer to include a restriction enzyme recognition site. This allows the previously harmful primer sequences to be specifically targeted and removed by the restriction enzyme, transforming the problem of primer contamination into a solution where the primer sequences serve as markers for their own removal.
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 the mapping efficiency and reduces errors in sequencing by exposing the internal DNA sequence, allowing for effective application of amplified DNA samples in next-generation sequencing, particularly with limited DNA samples, and reduces costs and time for sequencing processes.
Implementation Method 1
Digesting the amplified nucleic acid fragment with a restriction enzyme to remove the primer sequence thereby exposing the target sequence
Data Source
AI summary
A processing method to trim ends of DNA fragments, exposing the internal DNA part to give original DNA sequence information enabling application of next generation sequencing for DNA samples to be amplified by DOP-PCR or other primer dependent amplification methods. Specifically, nucleic acids are amplified using primers comprising a recognition site for a restriction enzyme, for example Bpml or Mmel. Primer sequences are removed by cleavage with the restriction enzyme.