LIFE09200604007 Tabish - Homi Bhabha National Institute

Materials and Methods
Method:
Restriction digestion of the PCR product with appropriate restriction enzyme
(Table 2) was done to analyse the genotype. 10 μl of PCR product out of 25 μl reaction
mixture was incubated with restriction enzyme at a concentration of 2 U/10 μl and
1/10 th volume of appropriate buffer in the final volume of 20 μl at 37 °C overnight.
Restriction digestion products were analysed on 2% agarose gel electrophoresis
described in section 8.2 along with 100 bp DNA ladder.
3.8.5 SNaPshot reaction:
SNaPshot assay developed on the principle of single base extension of primer
serves as a simple, sensitive, robust, time saving and specific approach to detect
multiple known SNPs in a pooled PCR product containing more than one gene. A
primer which anneals immediately adjacent to the SNP is extended by one base using a
fluorescently labelled ddNTP. Unlike PCR-RFLP, in SNaPshot assay the presence of
any restriction site for the detection of polymorphism is not mandatory. This method
further offers a direct visualization of the genotype of the SNP present in the expected
region on a DNA fragment by its novel four different fluorescently labeled ddNTPs
which give a coloured peak when incorporated in the SNP region on a
electropherogram after capillary electrophoresis. Thus, allowing SNP genotyping on a
large scale and in a short time.
Materials:
1. Amplified PCR product
2. PCR products
3. EXO-1 (10 U/μl) (USB)
4. SAP (1 U/μl) (USB)
5. SNaPshot ready reaction mix (provided in the kit) (Applied Biosystems)
6. Primers (Sigma-aldrich)
7. PCR buffer (10X)
8. Thermalcycler (Tetrad2 by BIO-RAD)
9. Distilled water
10. Genetic analyzer (Applied Biosystems- Avant 3100/3500)
Method:
First the desired gene was amplified by PCR using gene specific primers as
described in section 8.3. SNaPshot assay was performed which consists of three steps
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