2C. Day 1: Exploration of Gene analysis tools, primer designing and optimization (Lecuture cum Hands on Session) - bioinfokushwaha/Livestock_Genomics GitHub Wiki
Primer design
Polymerase Chain Reaction is a revolutionary technology that has greatly advanced the understanding of genes and genomes, and the bottleneck in this pursuit is designing quality primers. Therefore, this section discusses the key steps involved in primer designing and mentions the relevant software for each and every step. It is important to note the primers designing tutorial here is for gene specific primers alone (qPCR), not for gene cloning. Several bioinformatics tools are available to assist in primer designing for PCR like NCBI Primer-BLAST, Primer3, PrimerQuest, and Primer Premier. Here, NCBI Primer-BLAST is used to provide a hands on session for designing the primers.
1. Retrieve the FASTA sequence of the desired organism from NCBI/Gene
2. Use NCBI Primer BLAST to design primers against this sequence, and set the amplicon size within 500bp and hit with default parameters.
3. Pick the top primers from the results, amplicons with a size of approximately 100-250bp are ideal.
4. Use Oligocalc to assess primer properties and ensure no hairpin loops or secondary structures are formed.
5. Use NCBI Primer BLAST / Nucleotide BLAST to verify the primer specificity against the target sequence
Practice Exercise
Design gene specific primers for heat shock protein HSPA1L
Gene ID - 540190