Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.
Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and e...
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doaj-abdc59366c454ec1a5adca9aba5d96232021-03-03T21:35:17ZengPublic Library of Science (PLoS)PLoS ONE1932-62032020-01-01153e022878910.1371/journal.pone.0228789Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10.Vera HashemAnjana TiwariBrittani BewickHelio A G TeiveMariana MoscovichBirgitt SchüeleKhalaf BusharaMatt BowerAstrid RasmussenYu-Chih TsaiTyson ClarkKaren McFarlandTetsuo AshizawaLarge expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies.https://doi.org/10.1371/journal.pone.0228789 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Vera Hashem Anjana Tiwari Brittani Bewick Helio A G Teive Mariana Moscovich Birgitt Schüele Khalaf Bushara Matt Bower Astrid Rasmussen Yu-Chih Tsai Tyson Clark Karen McFarland Tetsuo Ashizawa |
spellingShingle |
Vera Hashem Anjana Tiwari Brittani Bewick Helio A G Teive Mariana Moscovich Birgitt Schüele Khalaf Bushara Matt Bower Astrid Rasmussen Yu-Chih Tsai Tyson Clark Karen McFarland Tetsuo Ashizawa Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. PLoS ONE |
author_facet |
Vera Hashem Anjana Tiwari Brittani Bewick Helio A G Teive Mariana Moscovich Birgitt Schüele Khalaf Bushara Matt Bower Astrid Rasmussen Yu-Chih Tsai Tyson Clark Karen McFarland Tetsuo Ashizawa |
author_sort |
Vera Hashem |
title |
Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. |
title_short |
Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. |
title_full |
Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. |
title_fullStr |
Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. |
title_full_unstemmed |
Pulse-Field capillary electrophoresis of repeat-primed PCR amplicons for analysis of large repeats in Spinocerebellar Ataxia Type 10. |
title_sort |
pulse-field capillary electrophoresis of repeat-primed pcr amplicons for analysis of large repeats in spinocerebellar ataxia type 10. |
publisher |
Public Library of Science (PLoS) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2020-01-01 |
description |
Large expansions of microsatellite DNA cause several neurological diseases. In Spinocerebellar ataxia type 10 (SCA10), the repeat interruptions change disease phenotype; an (ATTCC)n or a (ATCCT)n/(ATCCC)n interruption within the (ATTCT)n repeat is associated with the robust phenotype of ataxia and epilepsy while mostly pure (ATTCT)n may have reduced penetrance. Large repeat expansions of SCA10, and many other microsatellite expansions, can exceed 10,000 base pairs (bp) in size. Conventional next generation sequencing (NGS) technologies are ineffective in determining internal sequence contents or size of these expanded repeats. Using repeat primed PCR (RP-PCR) in conjunction with a high-sensitivity pulsed-field capillary electrophoresis fragment analyzer (FEMTO-Pulse, Agilent, Santa Clara, CA) (RP-FEMTO hereafter), we successfully determined sequence content of large expansion repeats in genomic DNA of SCA10 patients and transformed yeast artificial chromosomes containing SCA10 repeats. This RP-FEMTO is a simple and economical methodology which could complement emerging NGS for very long sequence reads such as Single Molecule, Real-Time (SMRT) and nanopore sequencing technologies. |
url |
https://doi.org/10.1371/journal.pone.0228789 |
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