TmPrime

TmPrime designs oligonucleotide sets optimized for homologous melting temperatures to facilitate gene assembly using ligase chain reaction (LCR) and polymerase chain reaction (PCR).

Key Features:

• Dynamic Oligonucleotide Design: Divides long DNA sequences based on user-defined melting temperatures and dynamically adjusts oligonucleotide lengths to achieve homologous melting temperatures.
• Comprehensive Output: Outputs melting temperatures, oligonucleotide sequences, and assessments of potential secondary structures.
• Sequence Pooling and Fragmentation: Partitions long genes into smaller fragments and pools suitable for multi-pool assembly.
• Codon Optimization: Performs codon optimization to align synthetic gene codon usage with target host systems.
• Purity Estimation via Melting Curve Analysis: Uses melting curve analysis to estimate purity of assembled gene products as an alternative to agarose gel electrophoresis.

Scientific Applications:

• Gene design and synthesis: Designs and synthesizes genes such as green fluorescent protein fragment (GFPuv), human protein kinase B-2 (PKB2), and the promoter region of human calcium-binding protein A4 (S100A4).
• Real-time PCR assembly and efficiency comparison: Supports real-time PCR assembly workflows using LCGreen I and provides melting-curve-based comparisons of gene synthesis efficiency.

Methodology:

Input a DNA sequence and target melting temperatures; TmPrime divides sequences, optimizes oligonucleotide lengths to achieve homologous melting temperatures, evaluates potential secondary structures, generates oligonucleotide sets and pooled fragments for multi-pool assembly, and employs melting curve analysis for purity estimation.