UCLA researchers in the Department of Dentistry have established novel extraction and bioinformatics pipeline of ultrashort single-stranded cell-free DNA for the use in liquid biopsy diagnostics.
BACKGROUND: Liquid biopsy is a critical diagnostic tool for various human diseases. The rapid market growth of liquid biopsy is primarily driven by novel early cancer detection, monitoring, and treatment response assessment. Cancer research has uncovered new biomarkers including circulating tumor DNA which takes form as free single-stranded DNA molecule fragments. In particular, ultrashort single-stranded cell-free DNA (uscfDNA) derived from 167bp mononucleosomal cell-free DNA (mncfDNA) is the most promising for biomarker use. They are non-mutational and reflect the host’s response to disease development and complements that cancer detection sensitivity of mutational ctDNA. Detection of these DNA types opens up many opportunities for saliva samples as a noninvasive disease detection. However, its use as an early cancer diagnostic and prenatal disease tool is restricted by the small amounts of cfDNA that can isolated from traditional approaches. This can result in issues with library construction and produce limited screening accuracy. Novel biochemical and bioinformatic methodologies are needed to increase clinical sensitivity of cfDNA-based liquid biopsy.
INNOVATION: UCLA researchers developed a novel nucleic acid extraction technique designed to separate DNA of different molecular weights and footprints including uscfDNA. The isolated DNA can subsequently be used for next-generation sequencing (NGS) analysis. Using various silica filters, one can extract 25-75nt uscfDNA from 167bp mncfDNA with high specificity. Additionally, researchers have established a NGS pipeline to evaluate the methylation profile of uscfDNA that is useful for disease detection. This is accomplished using 5mC-protected adapters on single-stranded DNA prior to bisulfite conversion and its incorporation into the final library construction. Adapting the molecular biology and bioinformatic pipeline can improve clinical diagnostic accuracy of uscfDNA detection.
POTENTIAL APPLICATIONS:
- Clinical Diagnostic platform for isolation and analysis of single-stranded cell-free DNA
- Methodology and pipeline for molecular biology research
ADVANTAGES:
- Increased extraction efficiency and isolation specificity of ultra-short single-stranded cell free DNA (25-75 nt)
- Increased sensitivity to methylation patterns of cell-free DNA
DEVELOPMENT-TO-DATE: Successful demonstration of invention in human liquid biopsy has been shown
Related Papers (from the inventors only):
1. Cheng J, Swarup N, Li F, Kordi M, Lin CC, Yang SC, Huang WL, Aziz M, Kim Y, Chia D, Yeh YM, Wei F, Zheng D, Zhang L, Pellegrini M, Su WC, Wong DTW. Distinct Features of Plasma Ultrashort Single-Stranded Cell-Free DNA as Biomarkers for Lung Cancer Detection. Clin Chem. 2023. Epub 20230919. doi: 10.1093/clinchem/hvad131. PubMed PMID: 37725931.
2. Cheng JC, Swarup N, Morselli M, Huang WL, Aziz M, Caggiano C, Kordi M, Patel AA, Chia D, Kim Y, Li F, Wei F, Zaitlen N, Krysan K, Dubinett S, Pellegrini M, Wong DTW. Single-stranded pre-methylated 5mC adapters uncover the methylation profile of plasma ultrashort Single-stranded cell-free DNA. Nucleic Acids Research. 2024. Epub 20240527. doi: 10.1093/nar/gkae276. PubMed PMID: 38797520.
3. Swarup N, Cheng J, Choi I, Heo YJ, Kordi M, Aziz M, Arora A, Li F, Chia D, Wei F, Elashoff D, Zhang L, Kim S, Kim Y, Wong DTW. Multi-faceted attributes of salivary cell-free DNA as liquid biopsy biomarkers for gastric cancer detection. Biomark Res. 2023;11(1):90. Epub 20231010. doi: 10.1186/s40364-023-00524-2. PubMed PMID: 37817261; PMCID: PMC10566128.