Project Examples – Multiplexed Determination of SNP Genotypes

Objective:  To develop a high throughput means of simultaneously determining poly-allelic SNP genotypes from multiple SNPs in multiple genes.

Performance Specifications: In DNA samples purified from 24 patients, correlation > 95% of all patient genotypes (multiple alleles in multiple genes) between IBA’s genotyping method and DNA sequencing.

Proposed Timeline: 14 Weeks

Description: This project involves the simultaneous identification of multiple SNP alleles in multiple genes (two genes, 4 SNPs, 8 different alleles total) in patient DNA.  IBA employed the BioPlex™ multianalyte detection system to develop an allele-specific primer extension methodology for the detection of a specific allele for a specific SNP.

In Phase 1, a multiplexed PCR protocol was developed that allowed the simultaneous amplification of all SNP gene sequences of interest from purified patient DNA.  Additionally, the conditions of the allele-specific primer extension reaction for each allele of each SNP were developed and optimized. 

In Phase 2, 24 patients of unknown genotypes were analyzed in a blinded fashion utilizing the newly developed multiplexed allele-specific primer extension assay.  A total of 192 genotypic assignments (24 patients; 8 possible alleles in four genes) were made based on comparison of assay signals generated in allele-specific primer extension reactions generated from patient DNA possessing known genotypes. 

Results: A multiplexed PCR and allele-specific primer extension methodology was developed that allowed the simultaneous amplification and subsequent determination of SNP genotype in four different SNPs (eight possible alleles).

Only 1 ng of patient DNA was required to perform both the PCR and allele-specific primer extension reactions.

Of the 192 genotypic assignments made using DNA from 24 patients, correlation of allele-specific primer extension and DNA sequencing results was > 98.5%.

Project Completion: 10 Weeks