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Table 1. Summary of recommendations for PCR primer design
Design Stage
Item
Recommendation
Reason
Preparation for design
Inclusivity database
Database and literature research on target sequences Full-length genomes or genes
Consensus design to conserved regions
Inclusivity database Exclusivity database
Fragments lead to design bias
near-neighbor sequences
Sequences likely to cause a false positive due to sequence relatedness or similar symptoms Reduce false positives from human genome, human refseq, human microbiome, soil microbes, etc. Needed for proper physical modeling and design
Background database
Gather contaminating genomes
Reaction conditions
Gather enzyme, buffer and salts, [NTPs], [Primers, Probes] Save all user settings and input, scoring terms and weights Capture all software parameters
Software input
Run software
Save input file
Allow for design to be reproduced, capture input for future meta-analysis using A.I. Allow for design to be reproduced, reduce input errors in subsequent runs Find signature regions that are conserved in the inclusivity and not found in exclusivity Not applicable to long genomes and to many genomes
Run from input file
software can be run from input file
Algorithm
k-mer analysis
Use to find signature regions using inclusivity and exclusivity databases Do not use for finding signature regions Align design regions of all members of inclusivity Target secondary structure, primer hybridization, primer dimers Scores for non-thermodynamic considerations Use thermodynamics-based scanning of primers against genomes Determine how well primers cover all members of inclusivity
Sequence alignments Sequence alignments
Use for reality check of consensus region only
Physical chemistry modeling
Naïve 2-state Tm is not sufficient
Heuristic scoring
Examples: G-quartets, low complexity, amplicon length, amplicon folding, etc. Check if primers are specific (PrimerBLAST, ThermoBLAST, ThermonucleotideBLAST) Determine mismatches and amount bound for all members of inclusivity Combine thermodynamic, heuristic, kmer, specificty, and other terms into a total score. Don't want control to interfere with desired target(s)
Specificity scoring
Coverage scoring
Total scoring
Weighted scoring equation
Add positive control
Check for compatibility of singplexes with the control gene
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