Short Tandem Repeat DNA Profiling Using Perylene-Oligonucleotide Fluorescence Assay.
Adrian H BustosElisa MartinyNadia Bom PedersenRohith Pavan ParvathaneniJonas HansenHanlee P JiKira AstakhovaPublished in: Analytical chemistry (2023)
We report an amplification-free genotyping method to determine the number of human short tandem repeats (STRs). DNA-based STR profiling is a robust method for genetic identification purposes such as forensics and biobanking and for identifying specific molecular subtypes of cancer. STR detection requires polymerase amplification, which introduces errors that obscure the correct genotype. We developed a new method that requires no polymerase. First, we synthesized perylene-nucleoside reagents and incorporated them into oligonucleotide probes that recognize five common human STRs. Using these probes and a bead-based hybridization approach, accurate STR detection was achieved in only 1.5 h, including DNA preparation steps, with up to a 1000-fold target DNA enrichment. This method was comparable to PCR-based assays. Using standard fluorometry, the limit of detection was 2.00 ± 0.07 pM for a given target. We used this assay to accurately identify STRs from 50 human subjects, achieving >98% consensus with sequencing data for STR genotyping.
Keyphrases
- single molecule
- nucleic acid
- endothelial cells
- circulating tumor
- high throughput
- label free
- cell free
- induced pluripotent stem cells
- single cell
- pluripotent stem cells
- small molecule
- loop mediated isothermal amplification
- high resolution
- machine learning
- big data
- electronic health record
- air pollution
- patient safety
- clinical practice
- simultaneous determination
- papillary thyroid
- heavy metals
- data analysis