Rolling Circle Amplification for Repetitive Nucleic Acid Sequences
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Solution Overview
Problem
Current nucleic acid amplification techniques, such as PCR, face challenges with amplifying targets containing short repeated sequences due to issues like stutter products and difficulties with palindromic or high GC content sequences, which introduce uncertainty in determining copy number and complicate accurate amplification.
Innovation Solution
The method involves using a circular nucleic acid template with a double-stranded central region and single-stranded hairpin end regions, where a primer is bound and extended by a strand-displacing polymerase, followed by endonuclease digestion and ligation to produce circular progeny nucleic acids, allowing for accurate amplification of repetitive sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If PCR amplification is used for targets containing short repeated sequences, then amplification can be achieved, but stutter products are generated that reduce measurement precision and introduce uncertainty in determining copy number
Solution Approach 1:
The patent segments the amplification process into distinct phases: initial PCR amplification followed by targeted rolling circle amplification (RCA). The RCA phase uses a circular template containing the amplified target region, with a primer binding site positioned outside the repeated sequence. This segmentation allows the problematic repeated sequence to be copied without generating stutter artifacts during the RCA phase, while still achieving the required amplification.
Solution Approach 2:
The patent introduces a circular nucleic acid template as an intermediary between the original linear target and the final amplified products. This circular template contains the target region flanked by unique sequences that serve as primer binding sites. The intermediary circular structure enables controlled replication that avoids the stutter problem while maintaining fidelity to the original repeated sequence.
2Productivity
If PCR amplification is used for palindromic sequences or sequences with extreme GC content, then amplification may be achieved, but the process becomes difficult and sometimes impossible
Solution Approach 1:
The patent inverts the traditional amplification approach by using rolling circle amplification instead of PCR. In RCA, a single primer binds to the circular template and the polymerase continuously synthesizes DNA around the circle, displacing the newly synthesized strand. This inversion of the amplification mechanism avoids the need for thermal cycling and primer annealing to difficult sequences, enabling amplification of palindromic and high GC content regions that are problematic for PCR.
Solution Approach 2:
The patent changes the fundamental parameters of the amplification process: replacing thermal cycling with isothermal conditions, using strand-displacing polymerase instead of standard polymerase, and using a circular template instead of linear DNA. These parameter changes collectively overcome the difficulties associated with amplifying challenging sequences by eliminating the steps where such sequences cause problems.
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 consistent and accurate amplification of target regions with repetitive sequences, reducing artifacts and improving the determination of copy number, even in challenging sequences like palindromes and high GC content areas.
Implementation Method 1
polymerase-mediated template-directed primer extension of the first primer is performed with a polymerase comprising strand displacement activity
Implementation Method 2
The first product is cut with the first endonuclease, and at least one first product hairpin is released
Implementation Method 3
circularizing the at least one first product hairpin comprises ligating a first hairpin adapter to the first product hairpin
Data Source
AI summary
Methods for amplifying a desired target region of a nucleic acid through rolling circle amplification with a strand-displacing polymerase are provided. Concatameric hairpin products are resolved with endonuclease digestion, and the resulting amplified product hairpins or fragments can be circularized and employed as templates in a subsequent round of amplification. The methods are effective for targeted amplification of even highly repetitive sequences. Compositions, kits, and systems related to or useful in the methods are also described.