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primer-basics

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Designs and ranks PCR primer pairs for a target template with primer3-py (design_primers), returning pairs with nearest-neighbor Tm, GC, product size, and complementarity scores. Covers why primer3 is a LOCAL weighted-penalty minimizer over the single template supplied (so PRIMER_PAIR_0 is the lowest-penalty pair under the given bounds, never a genome-specificity guarantee), why Tm is a salt/concentration-dependent SantaLucia prediction not a fixed property, why the two primers must be Tm-matched, the seq_args/global_args tag semantics (SEQUENCE_TARGET/INCLUDED/EXCLUDED/OVERLAP_JUNCTION/FORCE_*, 0-based [start,length]), 3'-end and GC-clamp mechanism, 5'-tail handling, masking SNPs under 3' ends, and diagnosing zero-pair runs. Use when designing standard PCR, cloning, genotyping, or sequencing primers, flanking a target, or screening pairs by Tm/size/GC. Genome off-target checking is primer-specificity; dimers/hairpins primer-validation; qPCR and probes qpcr-primers.

When to use

• designing standard PCR primers
• cloning primers with 5' tails
• genotyping primers flanking a target
• single sequencing primers
• screening pairs by Tm, size, and GC
• ranking primer pairs by weighted penalty

When not to use

• genome off-target checking
• primer-specificity
• dimer and hairpin validation
• primer-validation
• qPCR primers and probes
• qpcr-primers
• degenerate or consensus primers
• long-range PCR amplicons over 3-5 kb

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