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Cycling probe technology
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<h2 id="probe">Probe</h2> <p>Cycling probe technology makes use of a chimeric <a href="/facts/Hybridization_probe/j3pu1L7E">nucleic acid probe</a> to detect the presence of a particular <a href="/facts/DNA/nB89Iauz">DNA</a> sequence. The chimeric probe consists of an <a href="/facts/RNA/HxPeNfNj">RNA</a> segment sandwiched between two DNA segments. The RNA segment contains 4 contiguous <a href="/facts/Purine/9ogLp0Wc">purine</a> nucleotides. The probes should be less than 30 nucleotides in length and designed to minimize intra-probe and inter-probe interactions.<a class="footnote-ref" id="fnref:3" href="#fn:3"><sup>3</sup></a> </p> <h2 id="process">Process</h2> <p>Cycling probe technology utilizes a cyclic, isothermal process that begins with the <a href="/facts/Nucleic_acid_hybridization/j77KBWPJ">hybridization</a> of the chimeric probe with the target DNA. Once hybridized, the probe becomes a suitable substrate for <a href="/facts/Ribonuclease_H/UKaLv8fh">RNase H</a>. RNase H, an <a href="/facts/Endonuclease/t9Y0d883">endonuclease</a>, cleaves the RNA portion of the probe, resulting in two chimeric fragments. The <a href="/facts/Nucleic_acid_thermodynamics/4YD7V7hM">melting temperature</a> (Tm) of the newly cleaved fragments is lower than the melting temperature of original probe. Because the CPT reaction is isothermally kept just above the melting point of the original probe, the cleaved fragments dissociate from the target DNA. Once dissociated, the target DNA is free to hybridize with a new probe, beginning the cycle again.<a class="footnote-ref" id="fnref:4" href="#fn:4"><sup>4</sup></a><a class="footnote-ref" id="fnref:5" href="#fn:5"><sup>5</sup></a> </p><p>After the fragments have been cleaved and dissociated, they become detectable. A common strategy for detecting the fragments involves <a href="/facts/Fluorescence_in_the_life_sciences/KohWmQoS">fluorescence</a>. With this method, a fluorescent marker is attached to the 5’ end of the probe and a <a href="/facts/Quenching_(fluorescence)/3xRv4yIQ">quencher</a> is attached to the 3’ end of the probe.<a class="footnote-ref" id="fnref:6" href="#fn:6"><sup>6</sup></a><a class="footnote-ref" id="fnref:7" href="#fn:7"><sup>7</sup></a> When RNase H cleaves the probe, the quencher and fluorescent marker separate, increasing the intensity of the fluorescent marker. Cleaved fragments can alternatively be detected via amplification (e.g., <a href="/facts/Polymerase_chain_reaction/G0jWzJy1">PCR</a>) or further modification to allow for other chemical means of detection.<a class="footnote-ref" id="fnref:8" href="#fn:8"><sup>8</sup></a> </p><p>When working with small concentrations of target DNA, the CPT protocol can be modified to increase specificity and efficiency. Increasing allotted time has been shown to improve probe cleavage efficiency.<a class="footnote-ref" id="fnref:9" href="#fn:9"><sup>9</sup></a> Both increasing RNase H concentrations and use of a probe that isn't prone to inter-probe and intra-probe interactions has been shown to increase specificity.<a class="footnote-ref" id="fnref:10" href="#fn:10"><sup>10</sup></a> </p> <h2 id="advantages">Advantages</h2> <p>Because cycling probe technology does not involve the amplification of target DNA, CPT has a lower risk of <a href="/facts/Polymerase_chain_reaction_optimization/BweXhHHH">cross contamination</a> than PCR.<a class="footnote-ref" id="fnref:11" href="#fn:11"><sup>11</sup></a><a class="footnote-ref" id="fnref:12" href="#fn:12"><sup>12</sup></a> In addition, CPT is faster than PCR<a class="footnote-ref" id="fnref:13" href="#fn:13"><sup>13</sup></a> and doesn't require a specialized <a href="/facts/Thermal_cycler/9Y3ep6j4">thermocycler</a>. CPT also does not require running CPT products on a <a href="/facts/Gel_electrophoresis/9J3GrsFk">gel</a>. </p> <h2 id="disadvantages">Disadvantages</h2> <p>CPT requires specialized chimeric probes, making CPT assays more expensive than PCR.<a class="footnote-ref" id="fnref:14" href="#fn:14"><sup>14</sup></a> Because CPT probes are so specific, a new probe must be designed for each unique assay, further increasing cost. Clinical implementation is hampered financially, but it is also limited by the possibility of samples containing nonspecific <a href="/facts/RNAse/Fod4qUu5">RNases</a> other than RNase H.<a class="footnote-ref" id="fnref:15" href="#fn:15"><sup>15</sup></a> </p> <h2 id="applications">Applications</h2> <p>CPT can be used to detect specific DNA sequences and by extension specific <a href="/facts/Genotype/MlzK1Mfz">genotypes</a>. For example, CPT can be used to distinguish <a href="/facts/Genetically_modified_organism/6emCmpsc">GMO</a> produce from non-GMO produce.<a class="footnote-ref" id="fnref:16" href="#fn:16"><sup>16</sup></a> Clinically, CPT can be used as an alternative to cell culturing in order to detect <a href="/facts/Antimicrobial_resistance/jsbAywgx">antibacterial resistance</a> of a pathogen.<a class="footnote-ref" id="fnref:17" href="#fn:17"><sup>17</sup></a> </p><p>CPT, at its core, detects whether a specific sequence is present in a sample. But because cleaved probes accumulate following <a href="/facts/Rate_equation/VK18zUhN">linear rate kinetics</a>, the amount of target DNA can be quantified. Consequently, CPT has been used to quantify the number of non-coding repeats in organisms.<a class="footnote-ref" id="fnref:18" href="#fn:18"><sup>18</sup></a> </p><p>CPT can be used in conjunction with other technologies, like <a href="/facts/Molecular_beacon/InEra73Y">molecular beacons</a> and <a href="/facts/Real-time_polymerase_chain_reaction/jYLeLlQM">qPCR</a>.<a class="footnote-ref" id="fnref:19" href="#fn:19"><sup>19</sup></a> </p> <h2 id="references">References</h2> <ol> <li id="fn:1"><p>Bhatt R, Scott B, Whitney S, Bryan RN, Cloney L, Lebedev A (1999). "Detection of nucleic acids by cycling probe technology on magnetic particles: high sensitivity and ease of separation". Nucleosides & Nucleotides. 18 (6–7): 1297–9. doi:10.1080/07328319908044696. PMID 10474219. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:1" class="footnote-back-ref">↩</a></p></li> <li id="fn:2"><p>Tang T, Badal MY, Ocvirk G, Lee WE, Bader DE, Bekkaoui F, Harrison DJ (February 2002). "Integrated microfluidic electrophoresis system for analysis of genetic materials using signal amplification methods". Analytical Chemistry. 74 (4): 725–33. doi:10.1021/ac010874j. PMID 11866051. <a href="/wiki/Doi_(identifier)" target="_blank">/wiki/Doi_(identifier)</a> <a href="#fnref:2" class="footnote-back-ref">↩</a></p></li> <li id="fn:3"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:3" class="footnote-back-ref">↩</a></p></li> <li id="fn:4"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:4" class="footnote-back-ref">↩</a></p></li> <li id="fn:5"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:5" class="footnote-back-ref">↩</a></p></li> <li id="fn:6"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:6" class="footnote-back-ref">↩</a></p></li> <li id="fn:7"><p>Buh Gasparic M, Cankar K, Zel J, Gruden K (March 2008). "Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms". BMC Biotechnology. 8: 26. doi:10.1186/1472-6750-8-26. PMC 2322970. PMID 18325084. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970</a> <a href="#fnref:7" class="footnote-back-ref">↩</a></p></li> <li id="fn:8"><p>Wolcott MJ (October 1992). "Advances in nucleic acid-based detection methods". Clinical Microbiology Reviews. 5 (4): 370–86. doi:10.1128/cmr.5.4.370. PMC 358255. PMID 1423216. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC358255" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC358255</a> <a href="#fnref:8" class="footnote-back-ref">↩</a></p></li> <li id="fn:9"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:9" class="footnote-back-ref">↩</a></p></li> <li id="fn:10"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:10" class="footnote-back-ref">↩</a></p></li> <li id="fn:11"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:11" class="footnote-back-ref">↩</a></p></li> <li id="fn:12"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:12" class="footnote-back-ref">↩</a></p></li> <li id="fn:13"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:13" class="footnote-back-ref">↩</a></p></li> <li id="fn:14"><p>Buh Gasparic M, Cankar K, Zel J, Gruden K (March 2008). "Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms". BMC Biotechnology. 8: 26. doi:10.1186/1472-6750-8-26. PMC 2322970. PMID 18325084. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970</a> <a href="#fnref:14" class="footnote-back-ref">↩</a></p></li> <li id="fn:15"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:15" class="footnote-back-ref">↩</a></p></li> <li id="fn:16"><p>Buh Gasparic M, Cankar K, Zel J, Gruden K (March 2008). "Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms". BMC Biotechnology. 8: 26. doi:10.1186/1472-6750-8-26. PMC 2322970. PMID 18325084. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2322970</a> <a href="#fnref:16" class="footnote-back-ref">↩</a></p></li> <li id="fn:17"><p>Fong WK, Modrusan Z, McNevin JP, Marostenmaki J, Zin B, Bekkaoui F (July 2000). "Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology". Journal of Clinical Microbiology. 38 (7): 2525–9. doi:10.1128/JCM.38.7.2525-2529.2000. PMC 86959. PMID 10878037. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC86959</a> <a href="#fnref:17" class="footnote-back-ref">↩</a></p></li> <li id="fn:18"><p>Beggs ML, Cave MD, Marlowe C, Cloney L, Duck P, Eisenach KD (December 1996). "Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction". Journal of Clinical Microbiology. 34 (12): 2985–9. doi:10.1128/JCM.34.12.2985-2989.1996. PMC 229446. PMID 8940435. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC229446</a> <a href="#fnref:18" class="footnote-back-ref">↩</a></p></li> <li id="fn:19"><p>Jacroux T, Rieck DC, Cui R, Ouyang Y, Dong WJ (January 2013). "Enzymatic amplification of DNA/RNA hybrid molecular beacon signaling in nucleic acid detection". Analytical Biochemistry. 432 (2): 106–14. doi:10.1016/j.ab.2012.09.015. PMC 3522425. PMID 23000602. <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522425" target="_blank">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3522425</a> <a href="#fnref:19" class="footnote-back-ref">↩</a></p></li> </ol>