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Biochemically Programmable Isothermal PCR
Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for ado...
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| Published in: | Advanced science 2024-11, Vol.11 (41), p.e2404688-n/a |
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| Main Authors: | , , , |
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| container_issue | 41 |
| container_start_page | e2404688 |
| container_title | Advanced science |
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| creator | Kim, MinGin Ravisankar, Vijay Hassan, Yassin A. Ugaz, Victor M. |
| description | Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate‐limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate |
| doi_str_mv | 10.1002/advs.202404688 |
| format | article |
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Rapid isothermal PCR is achieved with 100% repeatability by manipulating the interplay between the reaction's biochemistry (via the amplicon GC content) and the lava‐lamp‐like microscale flow established inside a PCR tube whose upper and lower surfaces are isothermally maintained at annealing and denaturing temperatures.</description><identifier>ISSN: 2198-3844</identifier><identifier>EISSN: 2198-3844</identifier><identifier>DOI: 10.1002/advs.202404688</identifier><identifier>PMID: 39269276</identifier><language>eng</language><publisher>Germany: John Wiley & Sons, Inc</publisher><subject>biochemistry ; DNA - genetics ; DNA Primers - genetics ; Flow velocity ; Geometry ; nucleic acid analysis ; PCR ; point of care diagnostics ; Polymerase Chain Reaction - methods ; Reagents ; Reproducibility of Results ; Temperature ; Thermal cycling</subject><ispartof>Advanced science, 2024-11, Vol.11 (41), p.e2404688-n/a</ispartof><rights>2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH</rights><rights>2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.</rights><rights>2024. This work is published under http://creativecommons.org/licenses/by/4.0/ (the "License"). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><cites>FETCH-LOGICAL-c4155-22a9900e9ea932cfa95ad1f57ec52eca8ee69a793be4df2c671ba2a03421dd243</cites><orcidid>0000-0001-9361-9950</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.proquest.com/docview/3124285449?pq-origsite=primo$$EPDF$$P50$$Gproquest$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.proquest.com/docview/3124285449?pq-origsite=primo$$EHTML$$P50$$Gproquest$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,11562,25753,27924,27925,37012,37013,38516,43895,44590,46052,46476,53791,53793,74412,75126</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/39269276$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kim, MinGin</creatorcontrib><creatorcontrib>Ravisankar, Vijay</creatorcontrib><creatorcontrib>Hassan, Yassin A.</creatorcontrib><creatorcontrib>Ugaz, Victor M.</creatorcontrib><title>Biochemically Programmable Isothermal PCR</title><title>Advanced science</title><addtitle>Adv Sci (Weinh)</addtitle><description>Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate‐limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra‐fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection.
Rapid isothermal PCR is achieved with 100% repeatability by manipulating the interplay between the reaction's biochemistry (via the amplicon GC content) and the lava‐lamp‐like microscale flow established inside a PCR tube whose upper and lower surfaces are isothermally maintained at annealing and denaturing temperatures.</description><subject>biochemistry</subject><subject>DNA - genetics</subject><subject>DNA Primers - genetics</subject><subject>Flow velocity</subject><subject>Geometry</subject><subject>nucleic acid analysis</subject><subject>PCR</subject><subject>point of care diagnostics</subject><subject>Polymerase Chain Reaction - methods</subject><subject>Reagents</subject><subject>Reproducibility of Results</subject><subject>Temperature</subject><subject>Thermal cycling</subject><issn>2198-3844</issn><issn>2198-3844</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2024</creationdate><recordtype>article</recordtype><sourceid>24P</sourceid><sourceid>COVID</sourceid><sourceid>PIMPY</sourceid><sourceid>DOA</sourceid><recordid>eNqFkU1vEzEQhlcIRKvSK0cUiQscEuyxvfacUAlfkSpR8XW1Jt7ZZCNv3NpJUf49G1KilgsnW57Hj2bmrarnUkykEPCGmtsyAQFa6Nq5R9UpSHRj5bR-fO9-Up2XshJCSKOslu5pdaIQagRbn1av33UpLLnvAsW4G13ltMjU9zSPPJqVtFly7imOrqZfn1VPWoqFz-_Os-rHxw_fp5_Hl18-zaYXl-OgpTFjAEIUgpEJFYSW0FAjW2M5GOBAjrlGsqjmrJsWQm3lnICE0iCbBrQ6q2YHb5No5a9z11Pe-USd__OQ8sJT3nQhsjeDGhFV6wB0qBnRIkndhFpio8EOrrcH1_V23nMTeL3JFB9IH1bW3dIv0q2Xw65cbffdvLoz5HSz5bLxfVcCx0hrTtvilRTagEWDA_ryH3SVtnk97GqgQIMzWu-pyYEKOZWSuT12I4Xfp-r3qfpjqsOHF_dnOOJ_MxwAfQB-dZF3_9H5i_c_v6ERRv0G7_esCg</recordid><startdate>20241101</startdate><enddate>20241101</enddate><creator>Kim, MinGin</creator><creator>Ravisankar, Vijay</creator><creator>Hassan, Yassin A.</creator><creator>Ugaz, Victor M.</creator><general>John Wiley & Sons, Inc</general><general>John Wiley and Sons Inc</general><general>Wiley</general><scope>24P</scope><scope>WIN</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>3V.</scope><scope>7XB</scope><scope>88I</scope><scope>8FK</scope><scope>8G5</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BENPR</scope><scope>CCPQU</scope><scope>COVID</scope><scope>DWQXO</scope><scope>GNUQQ</scope><scope>GUQSH</scope><scope>HCIFZ</scope><scope>M2O</scope><scope>M2P</scope><scope>MBDVC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>Q9U</scope><scope>7X8</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0001-9361-9950</orcidid></search><sort><creationdate>20241101</creationdate><title>Biochemically Programmable Isothermal PCR</title><author>Kim, MinGin ; Ravisankar, Vijay ; Hassan, Yassin A. ; Ugaz, Victor M.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c4155-22a9900e9ea932cfa95ad1f57ec52eca8ee69a793be4df2c671ba2a03421dd243</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2024</creationdate><topic>biochemistry</topic><topic>DNA - genetics</topic><topic>DNA Primers - genetics</topic><topic>Flow velocity</topic><topic>Geometry</topic><topic>nucleic acid analysis</topic><topic>PCR</topic><topic>point of care diagnostics</topic><topic>Polymerase Chain Reaction - methods</topic><topic>Reagents</topic><topic>Reproducibility of Results</topic><topic>Temperature</topic><topic>Thermal cycling</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kim, MinGin</creatorcontrib><creatorcontrib>Ravisankar, Vijay</creatorcontrib><creatorcontrib>Hassan, Yassin A.</creatorcontrib><creatorcontrib>Ugaz, Victor M.</creatorcontrib><collection>Wiley Online Library Open Access</collection><collection>Wiley Open Access</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>Research Library (Alumni Edition)</collection><collection>ProQuest Central (Alumni)</collection><collection>ProQuest Central</collection><collection>ProQuest Central Essentials</collection><collection>AUTh Library subscriptions: ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Coronavirus Research Database</collection><collection>ProQuest Central</collection><collection>ProQuest Central Student</collection><collection>Research Library Prep</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Research Library</collection><collection>ProQuest Science Journals</collection><collection>Research Library (Corporate)</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><collection>ProQuest Central Basic</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>Advanced science</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kim, MinGin</au><au>Ravisankar, Vijay</au><au>Hassan, Yassin A.</au><au>Ugaz, Victor M.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Biochemically Programmable Isothermal PCR</atitle><jtitle>Advanced science</jtitle><addtitle>Adv Sci (Weinh)</addtitle><date>2024-11-01</date><risdate>2024</risdate><volume>11</volume><issue>41</issue><spage>e2404688</spage><epage>n/a</epage><pages>e2404688-n/a</pages><issn>2198-3844</issn><eissn>2198-3844</eissn><abstract>Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate‐limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported. Surprisingly, optimal performance depends on selecting primer sequences that replicate high GC content amplicons, contradicting established PCR primer design rules. This innovative thermocycling approach accelerates PCR to speeds rivaling ultra‐fast instruments, enabling rapid, repeatable isothermal DNA analysis across a range of targets relevant to diagnostics and pathogen detection.
Rapid isothermal PCR is achieved with 100% repeatability by manipulating the interplay between the reaction's biochemistry (via the amplicon GC content) and the lava‐lamp‐like microscale flow established inside a PCR tube whose upper and lower surfaces are isothermally maintained at annealing and denaturing temperatures.</abstract><cop>Germany</cop><pub>John Wiley & Sons, Inc</pub><pmid>39269276</pmid><doi>10.1002/advs.202404688</doi><tpages>9</tpages><orcidid>https://orcid.org/0000-0001-9361-9950</orcidid><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 2198-3844 |
| ispartof | Advanced science, 2024-11, Vol.11 (41), p.e2404688-n/a |
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| source | PubMed Central Free; Wiley Online Library Open Access; Publicly Available Content Database; Coronavirus Research Database |
| subjects | biochemistry DNA - genetics DNA Primers - genetics Flow velocity Geometry nucleic acid analysis PCR point of care diagnostics Polymerase Chain Reaction - methods Reagents Reproducibility of Results Temperature Thermal cycling |
| title | Biochemically Programmable Isothermal PCR |
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