The known DNA segments or candidate genes are sequenced by Sanger method, in order to obtain the full sequence of the target region to identify mutations or polymorphisms. Specific primers are designed to PCR amplify targeted region. Then purified PCR products are sequenced by Life Technologies’s BigDye sequencing kit (See figure 1). This sequencing approach is based on dideoxy chain-termination method. The sequencing reaction system contains four different dNTPs and a defined proportion of four different ddNTPs labeled with four different fluorescent dyes. In brief, once primers bind to the template, DNA polymerase extends the growing complementary chain by incorporating dNTPs and ddNTP through base-pair matching. Since ddNTPs are randomly added, so the extension reaction may be stopped at any position of the growing chain. Extension products with various length are labeled with fluorescence due to the incorporation of fluorescence labeled ddNTP. Then, these extension products in various length labeled with one of the four fluorescent dyes are separated by capillary electrophoresis in ABI sequencer (3130XL, 3730XL). Sequence information then can be interpreted by analysing the specific fluorescence signals from each extension fragments ordered in length.
Sample Requirement
Genomic DNA from various sources such as blood, tissue, diverse pathogenic microorganisms, and others.
Service Workflow
1. Primers design, synthesis and optimization.
2. Amplification and purification of target fragment.
3. PCR product sequencing.
4. Sequencing result interpretation, mutation and polymorphism loci calling.
5. SNP data processing, analysis, sequence diagram drawing to show mutation sites.
6. For polymorphic loci detection, LD map analysis, haplotype inference and tagSNP sites filtering are conducted.
Deliverables
1. Experiment record
2. Quality check report
