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Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies
Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatel...
Autores principales: | , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158611/ https://www.ncbi.nlm.nih.gov/pubmed/29635638 http://dx.doi.org/10.1093/nar/gky251 |
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author | Ladas, Ioannis Yu, Fangyan Leong, Ka Wai Fitarelli-Kiehl, Mariana Song, Chen Ashtaputre, Ravina Kulke, Matthew Mamon, Harvey Makrigiorgos, G Mike |
author_facet | Ladas, Ioannis Yu, Fangyan Leong, Ka Wai Fitarelli-Kiehl, Mariana Song, Chen Ashtaputre, Ravina Kulke, Matthew Mamon, Harvey Makrigiorgos, G Mike |
author_sort | Ladas, Ioannis |
collection | PubMed |
description | Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500–1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1–16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection. |
format | Online Article Text |
id | pubmed-6158611 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-61586112018-10-02 Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies Ladas, Ioannis Yu, Fangyan Leong, Ka Wai Fitarelli-Kiehl, Mariana Song, Chen Ashtaputre, Ravina Kulke, Matthew Mamon, Harvey Makrigiorgos, G Mike Nucleic Acids Res Methods Online Detection of microsatellite-instability in colonoscopy-obtained polyps, as well as in plasma-circulating DNA, is frequently confounded by sensitivity issues due to co-existing excessive amounts of wild-type DNA. While also an issue for point mutations, this is particularly problematic for microsatellite changes, due to the high false-positive artifacts generated by polymerase slippage (stutter-bands). Here, we describe a nuclease-based approach, NaME-PrO, that uses overlapping oligonucleotides to eliminate unaltered micro-satellites at the genomic DNA level, prior to PCR. By appropriate design of the overlapping oligonucleotides, NaME-PrO eliminates WT alleles in long single-base homopolymers ranging from 10 to 27 nucleotides in length, while sparing targets containing variable-length indels at any position within the homopolymer. We evaluated 5 MSI targets individually or simultaneously, NR27, NR21, NR24, BAT25 and BAT26 using DNA from cell-lines, biopsies and circulating-DNA from colorectal cancer patients. NaME-PrO enriched altered microsatellites and detected alterations down to 0.01% allelic-frequency using high-resolution-melting, improving detection sensitivity by 500–1000-fold relative to current HRM approaches. Capillary-electrophoresis also demonstrated enhanced sensitivity and enrichment of indels 1–16 bases long. We anticipate application of this highly-multiplex-able method either with standard 5-plex reactions in conjunction with HRM/capillary electrophoresis or massively-parallel-sequencing-based detection of MSI on numerous targets for sensitive MSI-detection. Oxford University Press 2018-07-06 2018-04-04 /pmc/articles/PMC6158611/ /pubmed/29635638 http://dx.doi.org/10.1093/nar/gky251 Text en © The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Ladas, Ioannis Yu, Fangyan Leong, Ka Wai Fitarelli-Kiehl, Mariana Song, Chen Ashtaputre, Ravina Kulke, Matthew Mamon, Harvey Makrigiorgos, G Mike Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title | Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title_full | Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title_fullStr | Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title_full_unstemmed | Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title_short | Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies |
title_sort | enhanced detection of microsatellite instability using pre-pcr elimination of wild-type dna homo-polymers in tissue and liquid biopsies |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158611/ https://www.ncbi.nlm.nih.gov/pubmed/29635638 http://dx.doi.org/10.1093/nar/gky251 |
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