For a considerable period, quantitative PCR (pPCR) has been utilized to accurately determine the number of target molecules. Yet this method relies on the generation of a ‘standard curve’ to quantify the target. Also, it involves monitoring of the reaction on a ‘real-time’ basis. An alternative to this is the ‘digital PCR’ (dPCR), which allows the PCR reaction to proceed to completion (i.e. endpoint PCR) and quantifies target molecules without requiring a reference curve or analyzing amplification curves (to obtain Ct values).
Originally, the above method was referred to as ‘single molecule’ or ‘limiting dilution’ PCR (Sykes et al., 1992; Morley, 2014). It was developed, in part, due to the ‘qualitative’ interest in cloning single target molecules by PCR to determine the sequence, ex. divergent strains of HIV (human immunodeficiency virus). Subsequently, with the introduction of the fluorescent technique (molecular beacon probe that fluoresces upon hybridizing to the PCR product) for monitoring the PCR result, the term ‘digital’ PCR was introduced (Vogelstein et al., 1999).
For ‘quantitative’ analysis, digital PCR can be used to determine the concentration of targets present in a minuscule amount. The sample is partitioned till it reaches the single-molecule level. For droplet PCR, samples are partitioned using water-oil emulsion media into tiny droplets (ranging from several thousand to millions), with each droplet capable of performing PCR. The results are obtained by monitoring each droplet (for fluorescence) and the number of droplets with positive results could be mathematically converted (based on Poisson distribution) to the concentration of the target in the sample stock. The alternate method is to employ chips containing hundreds of thousands of microchambers for PCR (instead of droplets). Instruments capable of supporting dPCR and/or qPCR reactions are available commercially with some capable of multiplexing, with the instruments becoming more affordable.
The principle underlying the dPCR method is based on Poisson distribution, a statistical treatment through which the probability for the occurrence of a given number of events can be predicted (assuming all events occur randomly and independently of one another). Briefly, for digital PCR, an “event” is defined as a droplet with a positive PCR result. Consider a case in which the ‘true value’ for the number of events (to be determined) is ‘X’. Then, the probability of obtaining a specific number of events (i.e. X-2, X-1, X, X+1, X+2, etc.) could be calculated based on Poisson distribution. Thus, the statistical analysis of the obtained results (number of droplets with a positive or negative results for dPCR) allows one to determine the target concentration in starting material.
Digital PCR can also be used to compare the frequency of different alleles (ex. mutant vs. wild-type). Regarding accuracy, dPCR could detect mutation frequencies reliably down to ~0.1%. This is comparable to ~0.1% mutation frequency detection (for BRAF V600E mutation) achieved at Bio-Synthesis, Inc. by utilizing BNA (bridged nucleic acid) incorporated clamp as well as a fluorescent probe for qPCR (https://www.biosyn.com/pdf/BRAF-Poster-Presented-at-AACR-2016.pdf). For detecting EGFR (epidermal growth factor receptor) mutation, 1 mutant in 22,000 wild-type molecules (~0.004%) detection limit by digital PCR was reported (Milbury et al., 2014). As for precision, a side-by-side comparison by Qiagen determined that dPCR exhibits lesser variability between independent assays than qPCR for target concentration down to ~50 copies/microliter.
Digital PCR can be used to quantitate the copy number of the target. Chronic myeloid leukemia (CML; ~15% of all adult leukemias) occurs due to the unregulated proliferation of white blood cells (myeloid cells). CML is associated with the occurrence of ‘Philadelphia chromosome’—chromosomal translocation between chromosomes 22 and 9, resulting in a fusion protein comprised of BCR and ABL (Melo et al. 1996). To block the function of the BCR-ABL protein, various chemical drugs inhibiting its tyrosine kinase activity (encoded by ABL) were developed, ex. Gleevec (a.k.a. Imatinib by Novartis) approved by FDA for treating CML in 2001. Despite the initial response to the above ‘cytostatic’ (arrests cell growth) drugs, the subsequent emergence of resistant CMLs necessitated the development of ‘second generation’ BCR-ABL inhibitors. The therapeutic response to BCR-ABL inhibitors is assessed by monitoring the level of BCR-ABL transcript in whole blood samples (<0.1% for ‘major molecular response’; <0.01% for ‘deep molecular response’) from treated CML patients. For this, real-time quantitative PCR has been utilized (Soverini et al., 2019). In 2019, FDA approved digital PCR for quantifying BCR-ABL mRNA. In its evaluation by Fred Hutchinson Cancer Research Center (USA), the authors noted additional advantages: less sensitive to PCR inhibitors due to endpoint counting, can increase sensitivity via sampling greater number of droplets (Shelton et al., 2022).
In the original publication by Vogelstein and colleagues (Johns Hopkins Oncology Center, USA), molecular beacons were used as probes to detect amplified products in the dPCR assay (Vogelstein et al., 1999). A molecular beacon contains a hairpin structure, which keeps its fluorophore quenched through energy transfer; upon hybridizing to the target sequence, the stem is disrupted, allowing it to fluoresce. In ‘QXDx BCR-ABL %IS Kit’ (BIO-RAD) developed to determine the level of BCR-ABL mRNA, a hydrolysis probe is used to detect BCR-ABL (at the breakpoint of translocation) as well as ABL. Hydrolysis probe contains fluorophore at 5’-end, whose fluorescence is blocked by a quencher at 3’end; upon degradation by 5’ to 3’ exonucleolytic activity of DNA polymerase during elongation, its fluorescence is restored. To increase the specificity and/or stability of the oligonucleotide probe for target recognition, modified nucleic acids can be incorporated such as ‘bridged nucleic acid’, ex. 2’,4’-BNANC containing an N-O bridged structure (Kim et al., 2015; Manning et al, 2015; Cromwell et al., 2018).
The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation. For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay. It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA) in addition to mRNA synthesis. A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as to conjugate to peptides or antibodies. It provides custom conjugation of small molecules such as chemical drugs, metabolites and labeled compounds with synthetic or natural polymers (enzymes, peptide, protein, oligonucleotide, antibody, dendrimer, nanoparticle, etc). It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides. To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade. It has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer. The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance. It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone. Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe). For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides.
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Shelton DN, Radich J, et al. Performance characteristics of the first Food and Drug Administration (FDA)-cleared digital droplet PCR (ddPCR) assay for BCR::ABL1 monitoring in chronic myelogenous leukemia. PLoS One. 17:e0265278 (2022). PMID: 35298544
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