Genotyping technology has undergone dramatic efficiency and speed changes because of ever-growing analysis needs for enormous numbers of polymorphisms in multiple samples. Many robust methods emerged with fast and efficient mechanisms for screening large populations for genetically linked traits and for cancer-related genes discovery. The NASR provides several different genotyping platforms.
Genotyping services include:
- Real-Time PCR-Based Assays
- Capillary Electrophoresis-Based Assays
- Illumina HiSeq 2000
Real-Time PCR-Based Assays (ABI 7900)
The Applied Biosystems 7900HT Sequence Detection Systems uses fluorescent-based PCR chemistries to provide qualitative detection of nucleic acid sequences by end-point analysis. An allelic discrimination assay is a multiplexed end-point assay that detects variants of a single nucleic acid sequence. The presence of two primer/probe pairs in each reaction allows genotyping of the two possible variants at the single-nucleic polymorphism (SNP) site in a target template sequence. TaqMan® SNP Genotyping Assays from Applied Biosystems provide a highly flexible technology for detection of polymorphisms within any genome. These TaqMan assays are routinely used for genotyping applications including screening, association, candidate region, candidate gene or fine-mapping studies.
Capillary Electrophoresis-Based Assays (ABI 3730)
The NASR routinely uses the ABI SNaPshot® Multiplex System for SNP analysis on one of the two 48-capillary Applied Biosystems 3730 DNA Analyzers. The SNaPshot Multiplex System is a primer extension-based method that allows multiplexing of up to 10 SNPs.
For SNP screening and confirmation, the PCR products are diluted and combined with the GeneScan™-120 LIZ® Size Standard to indicate the size of the labeled fragments. After the run, the GeneMapper® Software analyzes the data and generates allele calls.
Other common screening methods for cancers include Micro Satellite Instability (MSI) for colorectal cancer and Loss of Heterozygosity (LOH) for breast cancer using microsatellite markers.
Illumina HiSeq 2000
Genotyping in a selected segment of the genome can be achieved using Genomic DNA-seq protocol. This application is used for creating sequencing libraries from purified genomic DNA for subsequent analysis on the Illumina Cluster Station and HiSeq 2000.