Rolling Circle Amplification for Nucleic Acid Sequencing
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current nucleic acid sequencing techniques, such as sequencing-by-synthesis, are limited in the amount of sequence data they can reliably obtain, particularly for low-concentration starting materials and applications like SNP analysis or haplotyping, where additional sequence information is needed.
Innovation Solution
The method involves using oligonucleotides with specific primer sequences to form looped structures around target nucleic acid sequences, followed by rolling circle amplification to generate concatenated single-stranded nucleic acids, and CRISPR-Cas systems to cleave and release primers for amplification, enabling extended sequencing and detection of target nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If sequencing-by-synthesis techniques are used to obtain sequence data, then sequence data can be obtained for analysis, but the amount of sequence data that can be reliably obtained is limited to around 25-30 cycles of incorporation
Solution Approach 1:
The patent segments the sequencing process into two distinct phases: (1) initial sequencing-by-synthesis to generate reads with sufficient quality for realignment (25-30 cycles), and (2) rolling circle amplification to generate additional concatenated sequence data from the same templates. This segmentation allows each method to operate within its optimal performance range while collectively providing both reliable and extensive sequence data.
Solution Approach 2:
The patent performs preliminary sequencing-by-synthesis reactions to generate initial sequence reads that can be used for template realignment and identification. These preliminary reads serve as a foundation for subsequent rolling circle amplification, ensuring that the amplification process targets the correct templates while generating additional sequence data for variant analysis.
2Quantity of substance
If standard amplification methods are used on low concentration starting material, then amplification can occur, but sufficient sequence data may not be obtained for desired analysis
Solution Approach 1:
The patent implements continuous amplification through rolling circle amplification, where the DNA polymerase continuously synthesizes DNA around a circular template without termination. This continuous action generates long concatenated sequences containing multiple copies of the original template, dramatically increasing the amount of sequence data obtained from low-concentration starting material compared to discrete PCR cycles.
Solution Approach 2:
The patent changes the fundamental parameter of amplification architecture from linear PCR (discrete cycles with initiation and termination) to circular rolling circle amplification (continuous synthesis). This parameter change transforms the amplification process to generate much longer products with higher total sequence yield from the same starting material, enabling sufficient data for SNP analysis and haplotyping.
3Quantity of substance
If rolling circle amplification is used to generate concatenated nucleic acids, then amplification of low concentration targets is improved, but additional sequence information beyond 25-30 cycles may not be reliably obtained without proper template preparation
Solution Approach 1:
The patent performs preliminary sequencing-by-synthesis reactions to generate initial sequence reads that can be used for template realignment and identification before proceeding to rolling circle amplification. This preliminary action ensures that the templates selected for RCA are correctly identified and oriented, maintaining sequence data reliability while enabling extended amplification.
Solution Approach 2:
The patent uses the initial sequencing-by-synthesis reads as an intermediary to bridge the gap between low-concentration starting material and reliable sequence data. These intermediary reads serve dual purposes: they verify template integrity and provide the basis for designing the rolling circle amplification strategy, ensuring that the amplified data maintains reliability while increasing quantity.
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 allows for the reliable amplification and detection of target nucleic acids, especially at low concentrations, while retaining variant information, and facilitates next-generation sequencing and multiplexed analysis.
Implementation Method 1
contacting a target nucleic acid with an oligonucleotide such that the oligonucleotide binds to spaced-apart target binding sequences on the nucleic acid to form a looped structure
Implementation Method 2
extending a 3′ end of the oligonucleotide towards a 5′ end and across the target sequence
Implementation Method 3
ligating the extended 3′ end to the 5′ end of the oligonucleotide to form a closed loop
Implementation Method 4
using the closed loop as a template for rolling circle amplification to generate a concatenated single-stranded nucleic acid
Implementation Method 5
the first guide RNA contains a target-specific nucleotide region complementary to a first region of a target nucleic acid and the second guide RNA contains a target target-specific nucleotide region complementary to a second region of a target nucleic acid spaced apart from the first region; contacting the target nucleic acid with the system to form a complex to cleave within the first region and the second region to release an oligonucleotide
Implementation Method 6
annealing the oligonucleotide to a template; and amplifying the template using the annealed oligonucleotide as a primer
Data Source
AI summary
Nucleic acid amplification techniques are disclosed. Embodiments include generating concatenated nucleic acids using rolling circle amplification of templates, e.g., starting from a cDNA of a full-length mRNA or from synthetic templates, and sequencing and/or detecting the concatenated nucleic acids. In some embodiments, the technology disclosed includes amplification reactions that include CRISPR-Cas interactions that generate primers as a result of the CRISPR-Cas interactions, whereby primers are in turn used as part of detectable amplification reactions. The disclosed amplification techniques may use synthetic oligonucleotides or primers.