Assembly PCR Oligo Maker

Assembly PCR Oligo Maker designs oligodeoxynucleotides (ODNs) for two-step PCR-based assembly of long DNA molecules and for producing DNA templates suitable for in vitro transcription with T7 RNA polymerase.

Key Features:

• Automated design: Computes oligodeoxynucleotide sets required for a two-step PCR assembly of long DNA constructs.
• Flexible input handling: Accepts DNA or RNA sequences and converts RNA inputs into the corresponding DNA sequence.
• Oligonucleotide optimization: Generates oligonucleotide sequences optimized to achieve uniform melting temperature for consistent annealing conditions.
• Overlap verification: Checks for unintended regions of overlap outside desired priming sites to reduce non-specific amplification and primer-dimer formation.
• Output generation: Produces ready-to-use oligonucleotide sequences optimized for PCR-based assembly.
• Experimental validation: Design outputs have been experimentally validated, including synthesis of a 191-nucleotide DNA molecule using program-designed sequences.

Scientific Applications:

• Gene synthesis: Designing overlapping oligonucleotides for de novo construction of synthetic genes and large DNA constructs.
• RNA template production: Generating DNA templates for in vitro transcription with T7 RNA polymerase.
• Functional genomics: Constructing DNA sequences for functional studies and expression assays.
• Synthetic biology: Enabling assembly of complex genetic constructs for synthetic biology applications.

Methodology:

Accepts DNA or RNA input; converts RNA to corresponding DNA when needed; calculates oligodeoxynucleotides for a two-step assembly PCR while ensuring uniform melting temperatures and verifying overlap regions; outputs optimized oligonucleotide sequences.