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Sequence-specific preconcentration of a mutation prone KRAS fragment from plasma using ion-tagged oligonucleotides coupled to qPCR compatible magnetic ionic liquid solvents
- Source :
- Analytica Chimica Acta. 1068:1-10
- Publication Year :
- 2019
- Publisher :
- Elsevier BV, 2019.
-
Abstract
- Circulating tumor DNA (ctDNA) is a source of mutant DNA found in plasma and holds great promise in guiding cancer diagnostics, prognostics, and treatment. However, ctDNA fragments are challenging to detect in plasma due to their low abundance compared to wild-type DNA. In this study, a series of ion-tagged oligonucleotides (ITO) were synthesized using thiol-ene click chemistry and designed to selectively anneal target DNA. The ITO-DNA duplex was subsequently captured using a hydrophobic magnetic ionic liquid (MIL) as a liquid support. Extracted target DNA was quantified by adding the DNA-enriched MIL to the quantitative polymerase chain reaction (qPCR) buffer to streamline the extraction procedure. Clinically relevant concentrations of the mutation prone KRAS fragment, which has been linked to colorectal, lung, and bladder cancer, were preconcentrated using the ITO-MIL strategy allowing for enrichment factors as high as 19.49 ± 1.44 from pure water and 4.02 ± 0.50 from 10-fold diluted plasma after a 1 min extraction. Preconcentration could only be achieved when adding the ITO probe to the sample validating the selectivity of the ITO in the capture process. In addition, the amplification efficiency of qPCR was not affected when performing extractions from a diluted-plasma matrix demonstrating that the ITO-MIL approach coupled to direct-qPCR can be used to quantitate DNA from complex matrices. In comparison, commercially available steptavidin-coated magnetic beads were observed to lose selectivity when performing extractions from a 10-fold diluted plasma matrix. The selectivity of the ITO-MIL method, coupled with the ability to rapidly preconcentrate clinically relevant concentrations of target DNA from 10-fold diluted plasma, suggests that this method has the potential to be applied towards the extraction of ctDNA fragments from clinical samples.
- Subjects :
- Oligonucleotides
Ionic Liquids
02 engineering and technology
Polymerase Chain Reaction
01 natural sciences
Biochemistry
Analytical Chemistry
Proto-Oncogene Proteins p21(ras)
chemistry.chemical_compound
Humans
Environmental Chemistry
Spectroscopy
Magnetic ionic liquid
Chromatography
Oligonucleotide
010401 analytical chemistry
021001 nanoscience & nanotechnology
DNA extraction
0104 chemical sciences
Magnetic Fields
Real-time polymerase chain reaction
chemistry
Mutation
Ionic liquid
Solvents
Click chemistry
0210 nano-technology
Selectivity
DNA
Subjects
Details
- ISSN :
- 00032670
- Volume :
- 1068
- Database :
- OpenAIRE
- Journal :
- Analytica Chimica Acta
- Accession number :
- edsair.doi.dedup.....b0379bc56b28e51a4c89c7c15bc1f51c