INSILICO_PCR_&_PRIMER_DESIGN_methods.pptx

INSILICO_PCR_&_PRIMER_DESIGN_methods

1. In Silico PCR & Designing the Primer 1

2. WHAT IS INSILICO PCR In silico PCR is a computational method used to simulate and analyze Polymerase Chain Reaction (PCR) reactions using computer software and algorithms rather than physically conducting PCR experiments in a laboratory. It allows researchers to predict the outcomes of PCR reactions, including the expected amplicon size, location on the target DNA, and the specificity of the primers, all through computational analysis. In silico PCR is a valuable tool for experimental planning, primer design, and verifying the feasibility of PCR experiments without the need for physical laboratory work. 2

3. 3 WHY IS INSILICO PCR

4. 4 PROTOCOL FOR INSILICO PCR

5. 5 Collect Required Information: Determine the DNA sequence you want to analyze or amplify in silico. This could be a specific gene, genomic region, or any DNA sequence of interest. Prepare the primer sequences you intend to use for the in silico PCR.

6. 6 2. Choose an In Silico PCR Tool: You can use tools like UCSC Genome Browser, NCBI Primer-BLAST, or other softwares .

7. Pranavi Uppuluri 7

8. Pranavi Uppuluri 8

9. 9 3. Input Primer Sequences: Open the selected in silico PCR tool or website. Enter the primer sequences (forward and reverse) into the tool. Make sure the sequences are in the correct format (usually in 5' to 3' orientation).

10. Primer Design Using Software 10

11. Pranavi Uppuluri 11 INSILICO PCR TOOLS Performing in silico PCR can be done using various bioinformatics tools and software that are specifically designed for this purpose .

12. A number of primer design tools are available that can assist in PCR primer design for new and experienced users alike These tools may reduce the cost and time involved in experimentation by lowering the chances of failed experimentation Primer Design Using Software 12

13. Primer Designing

14. Primer A short single stranded oligonucleotide Binds to single stranded template molecule Acts as the starting point for complementary strand synthesis in 5’ 3’ Popular application: PCR application

15. Primer Selection Criteria Choose primers of 15 – 30 nucleotides in length. Longer primers provides sufficient specificity GC content of 40% - 60% Uniformly distributed More than 3 G/C nucleotide at the 3’ end should be avoided as nonspecific priming may occur Primers should end (3') in a G or C, or CG or GC: this prevents "breathing" of ends and increases efficiency of priming

16. Primer Selection Criteria Should not be self-complementary or complementary to other primers in the reaction to avoid primer dimer (self/cross) and hairpin Tm of flanking primers should not differ by over 5 °C, so the GC content should be chosen accordingly For degenerate primers at least 3 conservative nucleotides must be located at the primers’ 3’ end

17. 3. Primer3Plus 1. Primer3Plus 17

18. 3. Primer3Plus 1. Primer3Plus 18

19. 3. Primer3Plus 2. SnapGene Viewer 19

20. 3. Primer3Plus 2. SnapGene 20

21. 3. Primer3Plus 2. SnapGene 21

22. 3 . BiBiServ 22

23. 3 . BiBiServ 23

24. 4. PerlPrimer 4 . PerlPrimer 24

25. 4. PerlPrimer 4 . PerlPrimer 25

26. 4. PerlPrimer 4 . PerlPrimer 26

27. 5. PrimerQuest 5 . PrimerQuest 27

28. 5. PrimerQuest 5. PrimerQuest 28

29. 5. PrimerQuest 5 . PrimerQuest 29

30. 6. RealTimeDesign 6 . RealTimeDesign Software 30

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33. 33 4. Specify the Target Sequence: Provide the DNA sequence or genome where you want to perform the in silico PCR. This could be a specific accession number, gene name, or genomic location.

34. 34 5. Run the Analysis: It will simulate the PCR and give results, including: Expected amplicon size, Amplicon location on the target sequence, Primer specificity and any non-specific binding information.

35. 35 6. Interpret the Results: Review the results to ensure that the in silico PCR is specific to your target sequence and that the primer pairs are suitable for your experiment.

36. 36 7. Refine Primer Design (if necessary): If the in silico PCR results are not as expected or if there are issues with specificity, you may need to refine your primer design and repeat the analysis until you obtain satisfactory results .

37. Primer-BLAST ( NCBI) 37

38. Sequence Retrieval 38

39. Input Data 39

40. Primer Sets 40

41. Primer Sets 41

42. 42 8. Document the Results: Save the in silico PCR results, and document the parameters you used for the analysis for future reference.