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Molecular Tools for Biomarker Detection
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Molecular tools. Uppsala University, Science for Life Laboratory, SciLifeLab.ORCID iD: 0000-0002-5226-1427
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
Rolling circle amplification, padlock probe
National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-331745ISBN: 978-91-513-0114-3 (print)OAI: oai:DiVA.org:uu-331745DiVA: diva2:1150014
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
List of papers
1. A molecular approach for single molecule counting and rare mutation detection in blood plasma
Open this publication in new window or tab >>A molecular approach for single molecule counting and rare mutation detection in blood plasma
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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
Rolling circle amplification, cfDNA, single molecule, digital counting, rare mutation detection, PoC application
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331737 (URN)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
2. Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
Open this publication in new window or tab >>Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have recently developed a technology for localized sequence library preparation with rolling-circle amplification (RCA) as an approach for in situ sequencing. This method involves generation of clonally amplified and specially confined substrates for next-generation sequencing within the preserved context of cells and tissues. Our approach combines padlock probing, RCA, and sequencing-by-ligation chemistry that can resolve expression profiles of sets of genes and mutations in tissues without loss of histological context. Like other fluorescence-based assays, it can be hindered by high level of background fluorescence. To achieve high signal-to-noise ratios, we now describe a method to boost the amplification generated by RCA of padlock probes in situ by super RCA (sRCA). In this technique, a second padlock probe is hybridized, ligated and amplified on the first RCA product for enhanced, localized amplification. We describe and compare different sRCA strategies where gap-fill ligation was showed to be most efficient. The sRCA products co-localize and have comparable sizes as RCA products but they display at least two fold higher signal intensity. This increase in signal to noise also proved to result in two folds increase in the number of sRCA products detected. By combining sRCA with in situ sequencing for highly multiplex detection in tissue a four-time increase was seen. In summary, we demonstrate that sRCA can significantly increase the performance of padlock-based in situ sequencing for gene expression profiling of tissue sections, enabling detection of low abundant transcripts and the analysis of also highly auto-fluorescent samples. 

Keyword
in situ sequencing; super RCA (sRCA); tissue gene expression profiling, gap-fill padlock probe
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331741 (URN)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
3. Rolling Circle Amplification (RCA) Reporters – a new generic tool for the detection of DNA, RNA and proteins
Open this publication in new window or tab >>Rolling Circle Amplification (RCA) Reporters – a new generic tool for the detection of DNA, RNA and proteins
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Many methods to detect biomolecules with rolling circle amplification (RCA), for example padlock probes and proximity ligation assay (PLA), are tedious and includes several reaction steps combined with long incubation times. The present investigation evaluates a new tool to be included in the toolbox of RCA based detection methods that we refer to as RCA Reporter. The main idea with the RCA Reporter is to use pre-ligated circles protected from RCA initiation by almost entire hybridization to a protector molecule that unlocks through a well characterized and highly specific strand displacement process. The RCA Reporters are a generic tool that can be used to directly detect ssDNA or RNA in a sample, to detect proteins via coupling to proximity recognition using a pair of antibodies or to further amplify ongoing RCA reactions. The latter can be used to allow for efficient digital readout of single molecules via flow cytometry with the potential to develop simple and highly accurate molecular counting assays.

National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331743 (URN)
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
4. Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
Open this publication in new window or tab >>Elevated Levels of SOX10 in Serum from Vitiligo and Melanoma Patients, Analyzed by Proximity Ligation Assay
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 4, e0154214Article in journal (Refereed) Published
Abstract [en]

Background

The diagnosis of malignant melanoma currently relies on clinical inspection of the skin surface and on the histopathological status of the excised tumor. The serum marker S100B is used for prognostic estimates at later stages of the disease, but analyses are marred by false positives and inadequate sensitivity in predicting relapsing disorder.

Objectives

To investigate SOX10 as a potential biomarker for melanoma and vitiligo.

Methods

In this study we have applied proximity ligation assay (PLA) to detect the transcription factor SOX10 as a possible serum marker for melanoma. We studied a cohort of 110 melanoma patients. We further investigated a second cohort of 85 patients with vitiligo, which is a disease that also affects melanocytes.

Results

The specificity of the SOX10 assay in serum was high, with only 1% of healthy blood donors being positive. In contrast, elevated serum SOX10 was found with high frequency among vitiligo and melanoma patients. In patients with metastases, lack of SOX10 detection was associated with treatment benefit. In two responding patients, a change from SOX10 positivity to undetectable levels was seen before the response was evident clinically.

Conclusions

We show for the first time that SOX10 represents a promising new serum melanoma marker for detection of early stage disease, complementing the established S100B marker. Our findings imply that SOX10 can be used to monitor responses to treatment and to assess if the treatment is of benefit at stages earlier than what is possible radiologically.

Keyword
sox10 proximity ligation assay
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-289194 (URN)10.1371/journal.pone.0154214 (DOI)000374970600050 ()27110718 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 294409EU, FP7, Seventh Framework Programme, 316929Swedish Research Council
Available from: 2016-04-29 Created: 2016-04-29 Last updated: 2017-10-17Bibliographically approved

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