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.

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.

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. 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