Training Programme “Agri Bioinformatics Promotion Programme” DWR, Karnal(30th 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

1 Hybridization-based methods 1.1 Dynamic allele-specific hybridization 1.2 Molecular beacons 1.3 SNP microarrays2 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 assay3 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

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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.

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IUB/IUPAC Code of a SNP Alleles of a SNPMismatch 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

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)

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Tetra-ARM PCR

Ye, S. et al. Nucl. Acids Res. 2001 29:e88

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How Tetra ARM PCR works! Deliberate mismatch @ -2 from 3’ terminus If strong mismatch at 3’(G/A or C/T) then add ‘weak’

second mismatch (C/A or G/T) & vice versa If ‘medium’ mismatch (A/A, C/C, G/G or T/T) at 3’ end

will require a ‘medium second mismatch’ Primer length longer- 28 bases Inner- outer primer ratio (10 pM & 1 pM) PCR programme- Touch down Mg- usually higher (>1.5 mM)

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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. 704/13/23

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.

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Practical:1. Tetra arm PCR- for any SNP on this earth!-

Assignment- - , SNP genotyping2. Cleaver - To develop RFLP test for species

identification,

Assignment- Fish species signature dev

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