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Oligo Design for Multiplex PCR & High Throughput SNP Genotyping and Analysis

PrimerPlex is an efficient and sophisticated tool for designing oligos for multiplex assays. Multiplex assays facilitate amplification of multiple targets in a single reaction vessel, reducing both the time and cost of experimentation.
 

PrimerPlex designs oligos for multiplex PCR assays and for suspension array based systems such as Luminex 100, Luminex 200 and Bio-Plex 200. Such systems offer a versatile platform for multiplexed nucleic acid detection in a 96-well format. PrimerPlex designs specific capture probes for direct hybridization assays and primers for Allele Specific PCR Extension (ASPE) assays.

The multiplex assays are used for gene expression analysis (end point detection using gel electrophoresis) and for high throughput SNP applications such as genotyping, pathogen detection, strain typing, and haplotyping. To ensure specificity, the oligos are designed after avoiding the regions of homologies identified by a BLAST search. PrimerPlex then checks the oligos for cross reactivity and minimizes Tm mismatches to give you the best possible multiplex set. In the process, it analyzes millions of possible multiplex primer sets in a few seconds and presents a list of alternate sets to you
as well.

Design Options

Multiplex PCR Primer Design

PrimerPlex designs standard primers for a 30-plex multiplex PCR assay. It uses proprietary algorithms to design optimal primer sets under uniform reaction conditions for up to 30 targets. Primer sets are identified after screening all the primers in a pool and minimizing Tm mismatches. All the multiplex primer sets are analyzed for cross hybridization to ensure specific amplification and high signal strength.

Direct Hybridization Assays

PrimerPlex designs optimal allele specific capture probes for high throughput SNP genotyping assays. The probes are designed such that the mutations are at their centers to maximize differentiation. Each one of the probes designed is checked for its suitability in a multiplex reaction. For amplifying the templates, the program designs SNP flanking primers as well. This functionality makes PrimerPlex the only software product capable of designing probes for 100 individual targets for a multiplex assay.

ASPE Assays

Another approach to detect mutations is to use a solution-based sequence specific enzymatic reaction such as Allele Specific Primer Extension (ASPE) to determine the target genotype. PrimerPlex designs highly specific primers for multiplexed ASPE assays. The ASPE primers are designed such that the mutations lies at their 3' end. PrimerPlex also designs primers to amplify the target sequence for ASPE assays.

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