Ultra-sensitive detection of nucleic acid mutations for research and clinical use

What is SuperRCA?

SuperRCA is an ultra-sensitive and highly specific molecular amplification technology. It is used to detect very small amounts of DNA sequence variants, like cancer mutations, in patient tissue and blood samples.

SuperRCA offers effective detection of multiple targets simultaneously – so called multiplexing. The assay can be performed in most hematology laboratories with existing equipment, using well-established flow cytometry for read-out, enabling more accessible testing with shorter response times.

The convenient, non-intrusive liquid biopsy-format enables higher frequency of testing for improved patient monitoring and earlier detection of relapse.

In the clinic, SuperRCA can be used by the practicing clinician to monitor disease progress for adjusted treatment and personalized medicine. Our first application is in Acute Myeloid Leukemia (AML), offering single and multiplex testing for recurring tumor-specific mutations.

For researchers and pharmaceutical developers, our RUO kits offer a convenient and efficient toolbox for investigating numerous therapeutic areas to design companion diagnostics and advance personalized medicine.

SuperRCA offers a personal diagnostics platform for clinicians while providing an open learning system for researchers, partners and collaborations that allows new discoveries.

Ultra-sensitivity – detects 1 mutation in 100,000 wild types

SuperRCA can detect 1 mutation out of a 100 000 wild-type DNA molecules.

The unprecedented sensitivity of SuperRCA is achieved by two consecutive Rolling Circle Amplification (RCA) reactions. A first standard RCA step is followed directly by a subsequent “in situPadlock Probing and RCA step. As a result the target region is genotyped with high specificity, enumerated with higher precision.

SuperRCA method

An indepth introduction to SuperRCA

SuperRCA utilizes Rolling Circle Amplification (RCA) and Padlock probes in a novel way to achieve highly specific, ultra-sensitive detection of nucleic sequences. The method produces a relatively large, self-constrained structure – a SuperRCA structure – that can be directly analyzed by microscopy or automated using flow cytometry without the need for partitioning.

Flow cytometry with fluorescent labelling ensures that multiple targets can be analyzed simultaneously. As a result, the platform is extremely effective for patient-near, fast and cost-effective patient monitoring as well as companion diagnostics and pharmaceutical development .

DNA is extracted from the sample, either whole blood, bone marrow or tissue.

The DNA sequences of interest, known to be mutated in a patient’s malignant cells, are first enriched by a limited pre-PCR (~10 cycle) amplification.

The enriched sample then undergoes a ligase-mediated circularization of one strand.

The circularized strands containing the target region are then amplified by the first Rolling Circle Amplification  (RCA) step.

This is followed by Padlock Probe ligation and a highly-specific, second RCA step. During this step, the second RCA encircles the first RCA product to form large SuperRCA structures that can be analyze by flow cytometry.

The SuperRCA products can be scored as mutant- or wildtype-specific using fluorophore-labeled hybridization probes and recorded as individual, brightly fluorescent objects in a standard flow cytometer. For multiplex assays, analysis is similarly done by using multiple fluorophores and wavelengths.

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Why SuperRCA makes a difference

  • Unprecedented ultra-sensitivity of 1 mutation in 100 000 wild types due to the novel combination of the extremely high specificity of Padlock probes and RCA
  • Meets or exceeds currently available technologies like ddPCR, NGS and traditional liquid biopsy assays by 10-100 times
  • Outstanding performance on High GC% targets. Assessment of high GC content with the same sensitivity as low GC content – with minimum bias.
  • High multiplexity creates opportunities for effective research, monitoring therapies and drug resistance, sensitive multiplex detection of mutations for treatment selection (companion diagnostics), quantitative measurement for patient follow up and clinical phase drug development
  • Convenient and reliable analysis by well-established flow cytometry for speed by patient-near testing.
  • Other read-out and analysis methods including fluorescence microscopy
  • Speed and cost-effectiveness at high-throughput.
  • An integrated sample-to-answer system with robotic automation using standard lab equipment and well-established instrumentation, minimizes manual handling and is highly amenable to clinical practice
  • Currently available as research use only kit or as a customized service

What is Rolling Circle Amplification?

Rolling circle amplification (RCA) is an isothermal amplification method, which generates strands containing thousands of repeats complementary to the DNA circle that serve as the original template for the replication. The single-strand clustered amplification products can be labeled with fluorophores or chromogenic functional groups by oligonucleotides hybridization. RCA can be used to to magnify detection events locally into highly visible signals, in combination with the molecular tools that generate circular reaction products like in situ PLA, padlock probes, selector probes, PLAYR etc.

What are Padlock Probes?

A padlock probe is a short DNA oligonucleotide with segments at the 3’ and 5’ ends that are complementary to a target region. Upon hybridization, the two ends of the probe oriented in juxtaposition on the target template, leaving a nick site in the double-stranded structure. The nick site is sealed by a DNA ligase, and thereby the padlock probe is wound around and locked on the target strand. The DNA ligase activity is sensitive to base pair mismatches around the nick site, empowering the single base discrimination capacity of the padlock probes. The central part of the padlock probe is not target complementary and can harbor specific sequences serving different purposes, such as a detection probe hybridization site, sites for amplification primer hybridization, and capture probe binding site. Padlock probes have been used in many applications, for example for copy number variation analysis (CNV), single nucleotide polymorphism (SNP) analysis, gene expression profiling, alternative splicing analysis and pathogen detection.

DNA content in blood samples

The total DNA recovered from different tissues varies depending on type of sample, source, and health status of the patient. Solid biopsies from mammalian tissues typically yield 0.2-0.4 mg genomic DNA (gDNA)/mg tissue. The amount depends on how many nucleated cells the tissue contains per mg and fatty tissues such as brain, bone marrow or tissues with high levels of extracellular matrix such as connective tissues typically fall in the lower range.

Blood, a connective tissue with a dilute, liquid extracellular matrix, produces the lowest gDNA yields – typically between 15-50 ng gDNA/mg (or 15-50 mg gDNA/ml) with an assumed average of 35, corresponding to around 5 million nucleated white blood-cells/ml in healthy individuals.

  • A normal 10ml venous draw gives around 350mg gDNA, equivalent to around 100million haploid cells.

Even though blood is dilute with respect to gDNA, it is a readily available, non-invasive biological sample that is easily extracted from patients. Except for gDNA derived from nucleated cells, blood also contain circulating cell-free DNA (cfDNA) from recycled cells in the body. This pool of small, double stranded extracellular DNA fragments potentially reflects the genotypes of all the cells in the body and the concentration typically varies between 0-100 ng cfDNA/mlwith an average around 30 ng/ml for cancer patients. cfDNA is unstable with a reported half-life between 16 min to 2.5 h in circulation.

  • A normal 10ml venous draw gives around 300ng cfNDA, equivalent to around 91,000 haploid cells.
  • This means that in order to reliable sensitivity of 1:100,000, one would need 200,000 copies and hence 100,000 diplod cells, being 660ng cfDNA and hence around 22ml whole blood.

 

References:

  1. Elazezy M, Joosse SA. Techniques of using circulating tumor DNA as a liquid biopsy component in cancer management. Comput Struct Biotechnol J. 2018 Oct 9;16:370-378. doi: 10.1016/j.csbj.2018.10.002. PMID: 30364656; PMCID: PMC6197739.
  2. Anatoli Kustanovich, Ruth Schwartz, Tamar Peretz & Albert Grinshpun (2019) Life and death of circulating cell-free DNA, Cancer Biology & Therapy, 20:8, 1057-1067, doi: 10.1080/15384047.2019.1598759 
  3. Alborelli, I., Generali, D., Jermann, P. et al. Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study. Cell Death Dis 10, 534 (2019). https://doi.org/10.1038/s41419-019-1770-3