Other SNP Detection Protocols

The enormous task of associating various SNP panels to a specific disease presents tremendous challenge to technologies in SNP screening. Various assays have been developed for detection of known and unknown SNPs. Many commercial products are available as well. Some are relatively easy to do and low in cost. Others are developed for high volume screening at considerable cost. One must remember that there is hardly one protocol that meets all research needs. Very often, different protocols are utilized in a single core genotyping lab to provide flexibility, and accurate validation.

Majority of SNP genotyping assays can be separated into four groups based on molecular mechanisms.

Allele Specific Hybridization

Also known as ASO (allele specific oligonucleotide hybridization), this protocol relies on distinguishing between two DNA molecules differing by one base by hybridization. Fluorescence labeled PCR fragments are applied to immobilized oligonucleotides representing SNP sequences. After stringent hybridization and washing conditions, fluorescence intensity is measured for each SNP oligonucleotide.

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Primer Extension

In the single base extension approach, the target region is amplified by PCR followed by a single base sequencing reaction using a primer that anneals one base shy of the polymorphic site. Several detection methods have been described. One can label the primer and apply the extension products to gel electrophoresis. Or the single base extension product can be broken down into smaller pieces and measured by Mass Spectrometry. The most popular detection method involves fluorescence labeled, dideoxynucleotide terminators that stop the chain extension.

Primer Extensions with Terminators

Allele-Specific Primer Extensions

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Allele Specific Oligonucleotide Ligation

By designing oligonucleotides complementary to the target sequence, with the allele-specific base at its 3'-end or 5-'end, one can determine the genotype of the PCR amplified target sequence by determining whether an oligonucleotide complementary to the DNA sequencing adjoining the polymorphic site is ligated to the allele-specific oligonucleotide or not.

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Sequencing

Sequencing is the procedure of choice for SNP discovery. The most common forms of sequencing are based on primer extension using either a) dye-primers and unlabeled terminators or b) unlabeled primers and dye-terminators. The products of the reaction are then separated using electrophoresis using either capillary electrophoresis or slab gels.

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Determining what protocol is right for you

Before one decides on a particular protocol to use for SNP typing, one has to consider the following factors:

1. Scope of genotyping. This would include the # of SNPs to be screened, and # of samples to be tested, as well as how many genotyping projects are conducted at the same time. This will help determine if one needs a higher throughput format with relative ease to switch from one project to another.

2. Level of molecular biology expertise in the lab. Some assays are relatively simple to set up and perform, while others require considerable amount of experience in assay optimization and software support.

3. Funding for capital instrument. Capital investment for commercial SNP platforms can range from $30K to over $500K. Typically expensive sample processing automation is included in the high capital investment.

4. Consumable cost. Cost for consumables (reagents, plates, and pipett tips, etc) range from $0.20 to $5.00 per genotyping. This may not sound like a lot. However, when the project involves screening 1000 SNPs in a population of 1000, for a total of 1,000,000 genotypings, it can cost up to $5 million for a single project.

For more information regarding various SNP genotyping protocols, refer to the latest publication from Dr. Pui-Yan Kwok.

Kwok Pui-Yan. Methods for Genotyping Single Nucleotide Polymorphisms. Annu. Rev. Genomics Hum. Genet. 2001. 2: 235-258. (view abstract)

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