Global Lehigh Strategic Initiatives (without descriptions)
Snp Genotyping Methodlogy Dwr 30 03 10
1. Training Programme “Agri Bioinformatics Promotion Programme” DWR, Karnal (30 th March, 2010) SNP genotyping experimental designing using bioinformatics tools Dinesh Kumar , B.Sc. Hons Zoo(BHU), M.Sc. Biotechnology(BHU), Ph.D. Biotechnology (BHU), PDF(USA) Senior Scientist(Biotechnology) National Bureau of Animal Genetic Resources, Karnal-132 001, Haryana, INDIA Email: dineshkumarbhu@gmail.com, dinesh@iastate.edu
2. 1 Hybridization-based methods 1.1 Dynamic allele-specific hybridization 1.2 Molecular beacons 1.3 SNP microarrays 2 Enzyme-based methods 2.1 Restriction fragment length polymorphism 2.2 PCR-based methods 2.3 Flap endonuclease 2.4 Primer extension 2.5 5’- nuclease 2.6 Oligonucleotide ligase assay 3 Other post-amplification methods based on physical properties of DNA 3.1 Single strand conformation polymorphism 3.2 Temperature gradient gel electrophoresis 3.3 Denaturing high performance liquid chromatography 3.4 High-Resolution Melting of the entire amplicon 3.5 SNPlex SNP Genotyping methods 03/30/10 Dinesh-DWR-Bioinformatics training-30-03-10
3. Genotyping refers to the process of determining the genotype of an individual by the use of biological assays . Current methods of doing this include PCR , DNA sequencing , ASO probes, and hybridization to DNA microarrays or beads. 03/30/10 Dinesh-DWR-Bioinformatics training-30-03-10
4. The mismatches at the 3' end and the second mismatches at the third or other position from the 3' end in an allele-specific primer (You et al., 2008) 03/30/10 Dinesh-DWR-Bioinformatics training-30-03-10 IUB/IUPAC Code of a SNP Alleles of a SNP Mismatch at the 3' end (forward/reverse) Mismatch strength at the 3' end Second mismatch at the third or other position from the 3' end R G/A G/T Weak G/A, T/C A/C Weak A/G, C/T Y T/C G/T Weak G/A, T/C A/C Weak A/G, C/T S G/C G/G Medium C/C, A/A, G/G, T/T C/C Medium C/C, A/A, G/G, T/T W A/T A/A Medium C/C, A/A, G/G, T/T T/T Medium C/C, A/A, G/G, T/T K G/T G/A Strong G/T, A/C T/C Strong T/G, C/A M A/C G/A Strong G/T, A/C T/C Strong T/G, C/A
5. Tetra-ARM PCR Ye, S. et al. Nucl. Acids Res. 2001 29:e88 03/30/10 Dinesh-DWR-Bioinformatics training-30-03-10
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7. How to design tetra-armPCR for SNP genotyping? Figure : The input sequence having SNP is pasted in FASTA format and position of SNP and allele type is defined in the box. Here for example the position of SNP is 560, and it has two alleles viz A & G. One can also define the primer size in terms of length and also the product size which has to be visualized in ordinary agarose gel. 03/30/10
8. Figure : The output file showing two sets of primers which can be used in a single PCR reaction for SNP genotyping. The GG allele will have two bands (199 and 366) and AA allele will have two bands(221 and 366) and heterozygous allele (AG) will have three bands(199,221 and 366). A simple 100 bp ladder in agarose gel will be good enough to genotype the SNP in any modest lab. 03/30/10 Dinesh-DWR-Bioinformatics training-30-03-10
