FLASH Method for Low Abundance Sequence Enrichment
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Solution Overview
Problem
Current methods for enriching low-abundance sequences in nucleic acid samples are inefficient, difficult to optimize, and limited in the number of sequences that can be enriched, particularly in complex libraries.
Innovation Solution
The FLASH method employs sequence-specific nucleases like CRISPR/Cas9 to cut specific sites in DNA libraries, followed by ligation of adapter sequences for amplification, allowing for the enrichment of target sequences through a single PCR step using primers specific to the adapters.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiplex PCR or hybridization to labeled oligonucleotides is used to enrich low-abundance sequences, then some sequences can be enriched, but the method becomes inefficient, difficult to optimize, expensive to optimize, and limited in the number of sequences that can be enriched
Solution Approach 1:
The method segments the enrichment process into distinct steps: (1) blocking all DNA ends with phosphatase treatment, (2) targeted cleavage by Cas9 at specific sequences of interest, (3) adapter ligation to exposed ends, and (4) PCR amplification using adapter-specific primers. This segmentation allows simultaneous enrichment of multiple sequences without the complexity of multiplex PCR or hybridization optimization
Solution Approach 2:
The patent introduces adapters as intermediary molecules that bridge the Cas9 cleavage sites and the PCR amplification step. These adapters serve as universal handles for amplification, allowing any cleaved fragment to be enriched through a single PCR reaction using adapter-specific primers, thereby enabling enrichment of unlimited numbers of sequences with constant efficiency
2Measurement precision
If random fragmentation is used, then all sequences are represented, but low-abundance sequences are not sufficiently detected
Solution Approach 1:
The method extracts and enriches specifically low-abundance target sequences from the complex library by using Cas9 to cleave only at sequences of interest. By blocking all ends first and then selectively cleaving target sequences, the method isolates low-abundance sequences from the overwhelming background of high-abundance sequences, achieving up to tenfold enrichment and dramatically improved detection sensitivity
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 significantly increases the detection of low-abundance sequences, such as antibiotic resistance genes, by up to tenfold compared to random fragmentation, enhancing the sensitivity and efficiency of sequencing applications.
Implementation Method 1
a sequence-specific nuclease, such as CRISPR/Cas9, to cut specific sites of interest in a DNA library
Implementation Method 2
digesting a end-blocked (e.g., phosphatase-treated) mixed nucleic acid sample with a plurality of reprogrammed nucleic acid-directed endonucleases that target sequences of interest
Implementation Method 3
the ends of the DNA molecules in the DNA source may be blocked, e.g., treated with a phosphatase or using another method prior to nuclease digestion
Implementation Method 4
the newly exposed ends of the DNA are then free to be ligated to specific adapter sequences that allow them to be amplified
Implementation Method 5
a single PCR step using only a pair of primers specific to the adaptors can therefore amplify hundreds, thousands, or possibly millions of different sequences
Data Source
AI summary
A method of sample analysis is provided. In some embodiments, the method comprises: (a) digesting a mixed nucleic acid sample with a plurality of reprogrammed nucleic acid-directed endonucleases that target sequences of interest to produce a digested sample, wherein at least some of the fragments in the digested sample comprise: (i) a sequence of interest and (ii) at least one ligatable end that has been generated by endonuclease cleavage; (b) enriching for fragments that contain the sequence of interest; and (c) analyzing the enriched fragments. Kits for performing the method are also provided.