Enhanced detection of microsatellite instability using pre-PCR elimination of wild-type DNA homo-polymers in tissue and liquid biopsies.

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.

• Authors: Ioannis Ladas, Fangyan Yu, Ka Wai Leong, Mariana Fitarelli-Kiehl, Chen Song, Ravina Ashtaputre, Matthew Kulke, Harvey Mamon, G Mike Makrigiorgos
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 46 Issue 12 Pg. e74 (07 06 2018) ISSN: 1362-4962 [Electronic] England
• PMID: 29635638 (Publication Type: Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Circulating Tumor DNA
• Oligonucleotide Probes
• DNA

Topics

• Artifacts
• Biopsy
• Cell Line, Tumor
• Circulating Tumor DNA (blood)
• Colonic Neoplasms (blood, genetics, pathology)
• DNA (chemistry)
• Electrophoresis, Capillary
• Humans
• INDEL Mutation
• Liquid Biopsy
• Microsatellite Instability
• Oligonucleotide Probes
• Polymerase Chain Reaction