Abstract
Accurate and rapid identification or confirmation of single nucleotide polymorphisms (SNPs), point mutations and other human genomic variation facilitates understanding the genetic basis of disease. We have developed a new methodology (called MENA (Mismatch EndoNuclease Array)) pairing DNA mismatch endonuclease enzymology with tiling microarray hybridization in order to genotype both known point mutations (such as SNPs) as well as identify previously undiscovered point mutations and small indels. We show that our assay can rapidly genotype known SNPs in a human genomic DNA sample with 99% accuracy, in addition to identifying novel point mutations and small indels with a false discovery rate as low as 10%. Our technology provides a platform for a variety of applications, including: (1) genotyping known SNPs as well as confirming newly discovered SNPs from whole genome sequencing analyses; (2) identifying novel point mutations and indels in any genomic region from any organism for which genome sequence information is available; and (3) screening panels of genes associated with particular diseases and disorders in patient samples to identify causative mutations. As a proof of principle for using MENA to discover novel mutations, we report identification of a novel allele of the beethoven (btv) gene in Drosophila, which encodes a ciliary cytoplasmic dynein motor protein important for auditory mechanosensation.
Highlights
Current methodologies to identify known single nucleotide polymorphisms (SNPs) include microarray analysis (either based on differential hybridization strategies or hybridization coupled with DNA polymerase activity, as in the Affymetrix (Santa Clara, CA, USA) and Illumina (San Diego, CA, USA) platforms, respectively), Taqman® assays (Roche Diagnostics, Indianapolis, IN, USA), MassARRAY® /iPLEX® (Agena Bioscience, San Diego, CA, USA) single-base extension after amplification, mismatch endonuclease-based detection followed by suitable separation methods, or DNA sequencing [1,2,3,4,5,6,7,8,9]
For a 30 m probe, 5 + 30 + 5 probes per strand and per SNP nucleotide are required for this design strategy, with 5 + 40 + 5 probes per strand per SNP nucleotide required for 40 mer probes, etc
Deletion in btv22 relative to a wild type (WT) sequence (A); this deletion maps to exon 22 of btv (red rectangle chromosome arm 2L (position; 17966613-17966617); rectangle in B; B, red arrow in C), which is located in chromosome and (D)
Summary
Current methodologies to identify known single nucleotide polymorphisms (SNPs) include microarray analysis (either based on differential hybridization strategies or hybridization coupled with DNA polymerase activity, as in the Affymetrix (Santa Clara, CA, USA) and Illumina (San Diego, CA, USA) platforms, respectively), Taqman® assays (Roche Diagnostics, Indianapolis, IN, USA), MassARRAY® /iPLEX® (Agena Bioscience, San Diego, CA, USA) single-base extension after amplification, mismatch endonuclease-based detection followed by suitable separation methods, or DNA sequencing (either directed or whole-genome, as in medical resequencing or generation sequencing strategies) [1,2,3,4,5,6,7,8,9]. Mismatch endonuclease strategies are only able to interrogate SNPs whose identity is already known. Most of these methods are not scalable to query thousands of SNPs at a time. We focused on the Surveyor® endonuclease CEL II which is part of the CEL nuclease family derived from celery
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