SNP genotyping

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  • Published date: February 6, 2016
  • Modified date: February 7, 2016
    • Ankara, Turkey

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1. Sanger Sequencing Analysis:

This is as well-known gold standard method for identification of any mutation or polymorphism in target genomic sequence. Process includes the fluorescent labelled ddNTP incorporation during singe strand synthesis and the products are read by using capillary electrophoresis device. The most efficient and maximum length is accepted to be 600 bases read.

You may prefer Sanger sequencing if your application is among the subjects below :

- Sequence analysis of unknown target genomic regions, De-novo sequencing, varification of NGS data, mitochondrial dna sequencing etc.
- HLA typing,
- Identification of unknown genomic variations related to a disease or protein aberrations
- Forensics
- Agriculture and Animal Breeding
- Identification of epigenetics related regions on DNA and methylation analysis
- Identification of microorganisms related pathogenic diseases or environmental conditions by 16S rRNA gene sequence analysis and metagenomics

2. Restriction Endonuclease Analysis (REA)

Type-2 Restrion Endonuclease enzymes bind on and cut double-stranded DNA sequences with a sequence specific manner. They first discovered as an enzyme of bacterial defence mechanism against phages and then widely accepted in genome engineering. Restriction enzymes are both used in gene cloning and SNP genotyping applications.

Sanger sequencing is required for finding unknown genomic variations at specific locations, however it is expensive and time-consuming for scanning of large number of samples for a specific point. In this case, after amplification of target sequence by PCR, variations can be identified by observation of PCR products as cut or un-cut by specific restriction enzymes.

3. Allele Specific PCR Analysis

Scanning of specific variations on samples can not be performed by Restriction Enzyme Assay because there is not enough identified type-2 endonucleases for all sequence possibilities. Additionally, same enzyme binding sequences may be present at very close to target variation. In this case REA wont work.

Fast SNP genotyping can also be performed by an alternative method called Allele-Specific PCR. This method uses specific primers of which 3'-end hybridizes on the polymorphic/mutant nucleotide. By making two PCR reactions with 2 forward primers and one cummon reverse primer, one can find the mutation by observing the PCR product lines on gel.

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Serhat Sevli
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