SNP Genotyping with HEFP

• Free of gels, sequencing and labeled primers
• Rapid detection of 96 samples in less than 4 minutes
• Miniaturize to low volumes and 384-well plates
• Automate with stackers or full integration
• Easy SNP calling software

Researchers can now quickly and accurately call Single Nucleotide Polymorphisms (SNPs) by combining commercially available* single base extension technology with Molecular Devices' proven High Efficiency Fluorescence Polarization (HEFP™) detection technology and convenient AlleleCaller™ data analysis software.

flexible labeling by primer extension

SNPs of interest are PCR-amplified from genomic DNA samples in a standard 96- or 384-well PCR microplate. The excess PCR primers and dNTPs are then removed. An internal primer that lies just upstream from the polymorphic site is subsequently added together with a mixture of fluorescently lableled ddNTPs. The ddNTP terminator that is incorporated into the primer is dependent upon the polymorphism.

rapid detection by HEFP with Analyst^®

After incorporation of the ddNTPs, microplates are then loaded onto Molecular Devices' Analyst* multi-mode detection system. HEFP (High Efficiency Fluorescence Polarization) enables discrimination between labeled molecules of different molecular weights. HEFP easily differentiates between the size of free labeled-ddNTPs and the oligonucleotides in which the labeled-ddNTPs are already incorporated. HEFP is typically used for bi-allelic genotyping, using two dyes (R110 and TAMRA) to differentiate between different bases. Thus HEFP differentiates between free and incorporated ddNTPs and ddNTPs labeled with different dyes, enabling direct detection of two SNP alleles in a single assay well (figure 1). In comparison to other genotyping technologies, HEFP enables convenient single base extension and detection in the same microplate. And HEFP with Analyst is rapid, with detection of 96 samples in less than four minutes and 384 samples in less than nine minutes.

		Figure 1. Principles of detecting SNPs with HEFP.

 

Analyst is available in two versions: Analyst HT, designed for integration into robotic systems, and Analyst GT with an integrated Stacker, for automated processing of up to 40 microplates. All Analyst systems incorporate SmartOptics™, a unique (patents issued and pending) combination of light sources, optics and detectors that optimizes performance based on the selected mode, including the HEFP mode. Analyst systems provide the highest fluorescence sensitivity available in a multi-mode microplate system and have broad applications beyond genotyping using their fluorescence intensity, time-resolved fluorescence, luminescence and absorbance modes.

Figure 2. Analyst HT and Analyst HT with stacker.

easy data analysis with AlleleCaller

The AlleleCaller software, exclusively for Analyst users, translates the fluorescence polarization values in the instrument reports directly to bi-allelic single nucleotide polymorphism base calls. The results are displayed in a text column format and as a scatter-plot. The user has the flexibility to manually reassign genotypes on the scatter plot, which will be automatically saved in the report text file. AlleleCaller employs a unique clustering algorithm that performs automatic base-calling on results obtained from up to 384 individuals on one SNP. Additionally, reports from multiple plates can be processed in a batch mode.

		Figure 3. AlleleCaller scatter plot.

 

For more information on SNP genotyping with HEFP, please request a datafile and bibliography. Visit our Analyst literature page for details.
 

*Analyst^® and AlleleCaller™ from Molecular Devices Corporation may be useful for the detection of genetic polymorphisms (genotyping) by the method of SNP-IT™. Orchid BioSciences Inc. of Princeton, NJ asserts that the SNP-IT method is covered by U.S. Patent #5,888,819; 6,004,744 and their foreign counterparts. These Molecular Devices products may not be used for genotyping by the method of SNP-IT unless a separate license for such use is obtained from Orchid BioSciences, Inc. or one of its authorized sub-licensees. No license to practice SNP-IT is granted through the sale of this product to the purchaser or the end user.