{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T09:15:31Z","timestamp":1754558131870,"version":"3.41.2"},"reference-count":24,"publisher":"Wiley","issue":"5","license":[{"start":{"date-parts":[[2022,5,24]],"date-time":"2022-05-24T00:00:00Z","timestamp":1653350400000},"content-version":"vor","delay-in-days":23,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Current Protocols"],"published-print":{"date-parts":[[2022,5]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:p>Fragile X syndrome and other fragile X\u2012associated disorders are caused by the full\u2010mutation (&gt;200 copies) and premutation (55 to 200 copies) expansion, respectively, of the CGG short tandem repeat in the <jats:italic>fragile X messenger ribonucleoprotein 1<\/jats:italic> (<jats:italic>FMR1<\/jats:italic>) gene. Clinical diagnostic laboratories use Southern blot analysis and polymerase chain reaction (PCR)\u2010based tests to detect and\/or size the <jats:italic>FMR1<\/jats:italic> CGG repeats. The development of sensitive and high\u2010throughput triplet\u2010primed PCR (TP\u2010PCR) assays has diminished the need to subject all samples to Southern blot analysis, which is both labor\u2010 and time\u2010intensive. In this article, we describe two direct TP\u2010PCR (dTP\u2010PCR) assays for the detection of <jats:italic>FMR1<\/jats:italic> CGG repeat expansions. We outline a protocol that is based on melting curve analysis of dTP\u2010PCR amplicons for a rapid and cost\u2010effective first\u2010tier screening and identification of individuals with premutation and full\u2010mutation expansions. We also describe a protocol that employs capillary electrophoresis to resolve the dTP\u2010PCR amplicon fragments and to estimate the repeat sizes of normal (5 to 44 copies), intermediate (45 to 54 copies), and premutation alleles, as well as to detect full mutations and determine the structure of the <jats:italic>FMR1<\/jats:italic> alleles. \u00a9 2022 The Authors. Current Protocols published by Wiley Periodicals LLC.<\/jats:p><jats:p><jats:bold>Basic Protocol 1<\/jats:bold>: Direct triplet\u2010primed PCR master mix preparation and amplification of the <jats:italic>FMR1<\/jats:italic> CGG repeat locus for melting curve analysis<\/jats:p><jats:p><jats:bold>Basic Protocol 2<\/jats:bold>: Melting curve analysis of direct triplet\u2010primed PCR amplicons on the Rotor\u2010Gene Q MD \u00d7 5plex high\u2010resolution melt platform<\/jats:p><jats:p><jats:bold>Alternate Protocol<\/jats:bold>: Melting curve analysis of direct triplet\u2010primed PCR amplicons on the LightCycler 480 system<\/jats:p><jats:p><jats:bold>Basic Protocol 3<\/jats:bold>: Generation of direct triplet\u2010primed PCR melting curve analysis profiles<\/jats:p><jats:p><jats:bold>Basic Protocol 4<\/jats:bold>: Direct triplet\u2010primed PCR master mix preparation and amplification of the <jats:italic>FMR1<\/jats:italic> CGG repeat locus for capillary electrophoresis<\/jats:p><jats:p><jats:bold>Basic Protocol 5<\/jats:bold>: Generation of control <jats:italic>FMR1<\/jats:italic> plasmids for direct triplet\u2010primed PCR melting curve analysis<\/jats:p><jats:p><jats:bold>Basic Protocol 6<\/jats:bold>: Sanger sequencing assay to verify <jats:italic>FMR1<\/jats:italic> CGG repeat size and structure of plasmid DNA controls<\/jats:p>","DOI":"10.1002\/cpz1.427","type":"journal-article","created":{"date-parts":[[2022,5,24]],"date-time":"2022-05-24T18:33:28Z","timestamp":1653417208000},"source":"Crossref","is-referenced-by-count":2,"title":["Triplet\u2010Primed PCR Assays for Accurate Screening of <i>FMR1<\/i> CGG Repeat Expansion and Genotype Verification"],"prefix":"10.1002","volume":"2","author":[{"given":"Indhu\u2010Shree","family":"Rajan\u2010Babu","sequence":"first","affiliation":[{"name":"Department of Pediatrics, Yong Loo Lin School of Medicine National University of Singapore  Singapore Singapore"},{"name":"Department of Medical Genetics University of British Columbia  Vancouver British Columbia Canada"},{"name":"Children's &amp; Women's Hospital  Vancouver British Columbia Canada"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mulias","family":"Lian","sequence":"additional","affiliation":[{"name":"Khoo Teck Puat\u2013National University Children's Medical Institute National University Hospital  Singapore Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samuel S.","family":"Chong","sequence":"additional","affiliation":[{"name":"Department of Pediatrics, Yong Loo Lin School of Medicine National University of Singapore  Singapore Singapore"},{"name":"Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine National University of Singapore  Singapore Singapore"},{"name":"Department of Laboratory Medicine National University Hospital  Singapore Singapore"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2022,5,24]]},"reference":[{"key":"e_1_2_11_2_1","doi-asserted-by":"publisher","DOI":"10.1542\/peds.2016\u20101159H"},{"key":"e_1_2_11_3_1","doi-asserted-by":"publisher","DOI":"10.1542\/peds.2008\u20102992"},{"key":"e_1_2_11_4_1","doi-asserted-by":"publisher","DOI":"10.2353\/jmoldx.2010.090227"},{"key":"e_1_2_11_5_1","doi-asserted-by":"publisher","DOI":"10.1097\/GIM.0b013e31820a780f"},{"key":"e_1_2_11_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/0092\u20108674(91)90283\u20105"},{"key":"e_1_2_11_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrdp.2017.65"},{"key":"e_1_2_11_8_1","doi-asserted-by":"publisher","DOI":"10.1097\/GIM.0b013e3181d0d40e"},{"volume-title":"GeneReviews","year":"1993","author":"Hunter J. 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