Genotyping

Core Facilities

The Genotyping Core is a resource that allows NIEHS investigators to perform genotyping and screening of candidate genes and gene regions. Lauranell Burch, Ph.D. manages the facility, which assists with single nucleotide polymorphism (SNP) selection, sample preparation, sample management, genotyping and re-sequencing. Although NIEHS intramural funds provide support for the core, costs for use will be negotiated with each intramural investigator. The Genotyping Core offers the following services:

• Oracle-based LIMS project management with barcode interface
• DNA isolation, DNA databasing, sample management and storage
• Mining of existing SNP databases using the SNPselector program
• Compilation of local SNP database resources for SNP projects
• Mouse tail genotyping
• SNP genotyping
• Resequencing

SNPselector

The SNPselector program, written and maintained by Genotyping Core facility staff, is a high-throughput program for selecting SNPs for genetic linkage or association studies based on SNP quality, linkage disequilibrium, SNP function, SNP regulatory potential, and whether SNPs reside within repeat regions. SNPselector will be used where appropriate to find known SNPs in a given gene or region.

SNP discovery in affected populations by reseqeuncing

The core is equipped with two ABI model 3730 48-capillary sequencers to sequence DNA utilizing dye terminator chemistry. The facility uses a BioMek FX robot for sample preparation at a 1/64 dye scale to produce high-throughput resequencing data at low cost.

In addition to sample management and sequencing, the Genotyping Core will perform upstream bioinformatics and downstream data analysis to support resequencing projects.

Singleplex Genotyping by TaqMan or 3’mismatch assays

Investigators who desire genotyping results from a small number of SNPs may have samples genotyped by TaqMan assay (Applied Biosystems) or by a 3’mismatch SNP genotyping protocol developed in the core facility.

Multiplex SNP genotyping (96, 384 and 1536-plex) by the Illumina method

Illumina’s GoldenGate™ assay uses an allele-specific extension reaction and universal PCR technology to multiplex up to 1,536 SNP loci. Illumina’s arrays have 30X average redundancy of each bead type, for robust signal and genotyping accuracy. The Genotyping Core Facility’s Illumina workstation consists of pre and post-amp workstations in separate rooms to avoid PCR contamination.

Mouse Tail Genotyping

Mouse tail DNA extraction is automated on the BioMek FX robot and genotyping is performed by melting curve analysis of PCR product containing SYBR green dye.

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