primertervezés

Primer design, or primertervezés, is the process of selecting oligonucleotide sequences that initiate DNA synthesis under defined conditions, most commonly for polymerase chain reaction (PCR). The objective is to produce a specific amplification product from a target sequence while minimizing non-specific products.

Key factors include primer length (typically 18–25 nucleotides), GC content (about 40–60%), and melting temperature (Tm) compatibility between primer pairs, usually around 50–65°C for conventional PCR and similar temperatures for each primer in a pair. Primers should avoid secondary structures such as hairpins and primer-dimers, and avoid complementary sequences at their 3' ends to reduce non-specific amplification. Amplicon size matters: for standard PCR, products are often several hundred base pairs; for quantitative PCR (qPCR), shorter amplicons of roughly 70–200 bp are preferred.

Specificity and coverage are critical. Primers should match only the intended target in the genome, which is typically assessed in silico using tools like BLAST. SNPs in primer-binding sites should be avoided when possible, and highly repetitive regions or extreme GC-rich regions may require alternative strategies. Degenerate primers may be used for variable sequences, and nested or multiplex designs can enhance sensitivity or enable multiple targets.

Workflow generally includes defining the target region, selecting design parameters, and using primer design software (e.g., Primer3, Primer-BLAST). Candidate primers are evaluated for Tm, 3' stability, and absence of secondary structures, followed by in silico specificity checks. After synthesis, empirical testing and optimization of PCR conditions (Mg2+, annealing temperature, primer concentrations) are performed.

Applications include diagnostic PCR, cloning, sequencing, genotyping, and gene expression analysis by qPCR. Limitations arise from sequence quality and complexity, which can affect specificity and efficiency.