User:Saroj Pandey/Notebook/SNP PCR optimization

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Objective

  • a PCR to detect the tasting SNP rs713598 (a specific PCR for the G/C variant of the taste receptor TAS2R38)

gDNA extraction from buccal cavity

  • DNA extracted from a taster and a non-taster and concentration measured [1]

PCR for gDNA amplification

  • required segments of gDNA amplified using Taq DNA polymerase and sent for sequencing [2]

SNP PCR

  • SNP PCR conducted using gDNA and PCR products as templates separately [3]

Sequencing

  • Sequencing results confirm genotype with phenotype [4]

grdient PCR (SNP)

  • gradient PCR using fixed forward and fixed backward G primers conducted for both taster and non-taster [5]

Allele Specific PCR and Multiplex PCR

  • PCR was run using 4 primers (2 outer and 2 allele specific inner) with gDNAs as templates [6]

PCR with different primer combinations

  • PCR was performed using different combinations of primers using gDNAs as templates [7]

gradient PCR for optimization

  • Correct primer combinations from previous result were again used for a gradient PCR to find out the optimal annealing temperature [8]

PCR with spit sample

  • PCRs were carried out using spit sampes instead of gDNA templates [9]

Allele Specific PCR

  • PTC taster and non-taster were successfully differentiated genotypically [10]

Final separation and gradient PCR

  • One step PCR was optimized to separate PTC taster and non-taster. A gradient PCR was also performed to identify optimal temperature for this PCR. [11]