Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.
Nucleic Acids Res
; 46(12): e74, 2018 07 06.
Article
en En
| MEDLINE
| ID: mdl-29635638
ABSTRACT
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.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Biopsia
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Reacción en Cadena de la Polimerasa
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Neoplasias del Colon
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Inestabilidad de Microsatélites
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Nucleic Acids Res
Año:
2018
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
ENGLAND
/
ESCOCIA
/
GB
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GREAT BRITAIN
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INGLATERRA
/
REINO UNIDO
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SCOTLAND
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UK
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UNITED KINGDOM