Design validated qPCR primer pairs for a given DNA sequence using Primer3.

Args: sequence: Target DNA sequence (ATCG only, min 100 bp). target_tm: Target melting temperature in °C (default: 60.0). product_size_range: Desired PCR product size range (e.g., '70-150'). num_pairs: Number of primer pairs to return (1-5, default: 3).

Returns: A JSON-formatted string with validated primer pairs or error information.

Design primers for cloning the entire sequence with optional 5' overhangs. Detects common restriction sites and suggests leaders for efficiency.

Args: sequence: Target DNA sequence (ORF/Fragment to amplify). fwd_overhang: Optional 5' overhang for forward primer (e.g., restriction site). rev_overhang: Optional 5' overhang for reverse primer. target_tm: Target melting temperature in °C (default: 60.0).

Returns: A JSON string with precise and suggested (leader-included) primer pairs.

Design a TaqMan probe for the region between two primers. Ensures probe Tm is higher than primers and follows standard rules (no 5' G).

Args: sequence: Target DNA sequence containing the primer binding sites. primer_fwd: Forward primer sequence (5'->3'). primer_rev: Reverse primer sequence (5'->3'). target_probe_tm: Target melting temperature in °C (default: 68.0).

Returns: A JSON string with the designed probe or error message.

Check if a primer sequence binds elsewhere in the genome using NCBI BLAST. Requires internet connection and may take 10-30 seconds.

Args: primer_sequence: The DNA sequence of the primer (15-30 bp).

Returns: A JSON string with specificity rating and top matches in nt database.

Analyze a set of primers for potential cross-reactivity (heterodimers). Useful for multiplex PCR or panel design.

Args: primers: A list of primer sequences to analyze.

Returns: A JSON string with identified risk interactions and compatibility score.

Design a set of non-conflicting primer pairs for multiple genes. Uses a greedy approach to minimize heterodimers between pairs.

Args: genes: A list of genes, each with 'name' and 'sequence' keys. target_tm: Target melting temperature for primers in °C (default: 60.0).

Returns: A JSON string with the selected non-conflicting panel and any failures.