This technology increases the fraction of clinically relevant, rare mutant alleles (DNA and/or RNA) to enable high sensitivity detection with common downstream methods such as capped (XNA or PNA) PCR, ddPCR, and sequencing.
Liquid biopsy is a simple, minimally invasive, rapidly developing diagnostic method to analyze cell-free nucleic acid fragments in body fluids to obtain critical information on patient health and disease status, enable personalized therapy, and replace invasive methods such as tissue biopsy.
The sensitivity of available tests is, however, insufficient for patients at early disease stage and for cancer screening. Detection of alleles that contain critical clinical information is challenged by their very low concentrations and sequence homologies with abundant wildtype nucleic acids. This invention comprises of a high efficiency assay that reduces the concentrations of wildtype nucleic acids, DNA and/or RNA, and increases the fraction of clinically-relevant mutant alleles (DNA and/or RNA) to enable their downstream detection by a variety of methods.
This invention utilizes DNA-guided Argonaute (Ago) endonuclease that cleaves guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of clinically-relevant rare alleles in a patient sample to enhance the sensitivity of downstream detection methods such as drop digital PCR, sequencing, and clamped (e.g., XNA and PNA) enzymatic amplification, enabling detection of low-frequency (0.01%) mutant alleles.
This invention has many advantages over competing technologies such as CRISPR Cas9-based, as Ago does not require targets to contain any specific (PAM-like) motifs; is a multi-turnover enzyme; cleaves ssDNA, dsDNA, and RNA targets in a single assay; is DNA-guided (less expensive and more stable than RNA guides), and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.
- Improves sensitivity of downstream detection and genotyping methods
- Capable of enriching both DNA and RNA alleles in the same assay
- Versatile: does not require targets to contain specific (PAM-like) motifs
- Turnover enzymes: reduced enzyme consumption
- Highly specific: resolves single nucleotide differences between sequences.
- Can be used as standalone kit for qualitative and quantitative assay to enrich rare sequences or integrated with existing downstream amplification such as capped (RT-) PCR/LAMP and/or sequencing.
- Amenable for incorporation into point of care devices
- Increase the effectiveness and sensitivity of liquid biopsies
- Enable detection of specific genetic materials (e.g., mutations associated with various types of cancers and fetal disorders; and strains of pathogens
Stage of Development:
- Reduction to practice
- In a proof of concept experiments, this invention enabled detection of cancer-related cell free alleles at fractions as low as 0.1% in blood samples from pancreatic cancer patients, in excellent agreement with tissue biopsy genotyping.
- Provisional Patent Application No. 62642984
- Related PCT Filed
- International Patent PCT/US2017/013403 (“WO2017123921A1”)