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A molecular approach for single molecule counting and rare mutation detection in blood plasma
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University. (molecular tools)ORCID iD: 0000-0002-5226-1427
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (molecular tools)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Problems in biology and medicine frequently require the ability to observe, evaluate, and count even extremely rare macromolecules directly in biological samples. Examples include the detection of mutant DNA or RNA molecules in plasma or distributed in tissues in tumor patients, and highly precise, digital enumeration of proteins and other molecules of interest in clinical specimens. We describe herein a general means to magnify detection signals from individual molecules to easily recorded levels via a highly specific process - super rolling circle amplification (sRCA). We demonstrate the ability of this technique to vastly enhance in situ detection, to count individual molecules by flow cytometry or using a mobile phone camera, and to enumerate tumor-specific sequence variants in plasma from patients at very high efficiency, with specificity adequate to detect single nucleotide mutant sequences among 100,000 copies of the normal sequence. 

Keyword [en]
Rolling circle amplification, cfDNA, single molecule, digital counting, rare mutation detection, PoC application
National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-331737OAI: oai:DiVA.org:uu-331737DiVA: diva2:1149999
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
In thesis
1. Molecular Tools for Biomarker Detection
Open this publication in new window or tab >>Molecular Tools for Biomarker Detection
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The advance of biological research promotes the emerging of new methods and solutions to answer the biological questions. This thesis describes several new molecular tools and their applications for the detection of genomic and proteomic information with extremely high sensitivity and specificity or simplify such detection procedures without compromising the performance.

In paper I, we described a general method namely super RCA, for highly specific counting of single DNA molecules. Individual products of a range of molecular detection reactions are magnified to Giga-Dalton levels that are easily detected for counting one by one, using methods such as low-magnification microscopy, flow cytometry, or using a mobile phone camera. The sRCA-flow cytometry readout presents extremely high counting precision and the assay’s coefficient of variation can be as low as 0.5%. sRCA-flow cytometry readout can be applied to detect the tumor mutations down to 1/100,000 in the circulating tumor cell-free DNA.

In paper II, we applied the super RCA method into the in situ sequencing protocol to enhance the amplified mRNA detection tags for better signal-to-noise ratios. The sRCA products co-localize with primary RCA products generated from the gene specific padlock probes and remain as a single individual object in during the sequencing step. The enhanced sRCA products is 100% brighter than regular RCA products and the detection efficiency at least doubled with preserved specificity using sRCA compared to standard RCA.

In paper III, we described a highly specific and efficient molecular switch mechanism namely RCA reporter. The switch will initiate the rolling circle amplification only in the presence of correct target sequences. The RCA reporter mechanism can be applied to recognize single stranded DNA sequences, mRNA sequences and sequences embedded in the RCA products.

In paper IV, we established the solid phase Proximity Ligation Assay against the SOX10 protein using poly clonal antibodies. Using this assay, we found elevated SOX10 in serum at high frequency among vitiligo and melanoma patients. While the healthy donors below the threshold.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1387
Keyword
Rolling circle amplification, padlock probe
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331745 (URN)978-91-513-0114-3 (ISBN)
Public defence
2017-12-08, BMC/A1:111a, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-11-14 Created: 2017-10-17 Last updated: 2017-11-14

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