- Synonyms: T7 Primer, T7 Promoter Sequence Primer, T7 Forward Primer
- The T7 promoter primer is a short, single-stranded oligonucleotide designed to anneal to the T7 promoter sequence, which serves as the binding site for T7 RNA polymerase.
- The canonical sequence of the T7 promoter primer is: 5′-TAATACGACTCACTATAGGG-3′
- This sequence corresponds to the conserved T7 RNA polymerase recognition site, ensuring high-efficiency transcription. The primer is typically 20 nucleotides long, though some variations may include additional bases downstream to enhance transcription efficiency.
- The melting temperature (Tm) of the standard 20-base T7 promoter primer is approximately 50–55°C, depending on specific reaction conditions and buffer composition. The GC content is around 45% (with 9 G/C bases and 11 A/T bases). The annealing temperature typically ranges from 48–55°C, depending on the PCR setup. For high-specificity applications, such as in vitro transcription (IVT) or sequencing, the annealing step is often optimized to 50–52°C.
- For general applications, primers purified by standard desalting are sufficient. However, for high-precision applications – such as IVT, mRNA synthesis, and sequencing – HPLC or PAGE purification is recommended to ensure maximum purity and performance.
Applications:
- The T7 promoter primer is versatile and widely used in several key applications in molecular biology, particularly those that involve transcription, amplification, and sequencing.
- In Vitro Transcription (IVT): One of the primary uses of the T7 promoter primer is in in vitro transcription (IVT). In this application, the primer is used to synthesize RNA from a DNA template containing the T7 promoter. When T7 RNA polymerase is added, it recognizes the T7 promoter sequence and initiates transcription, producing large quantities of RNA. This method is commonly applied in mRNA production, riboprobe synthesis, and RNA virus studies.
- PCR Amplification: The T7 promoter primer also serves as a forward primer for PCR amplification of sequences that contain the T7 promoter. This is particularly useful for generating DNA templates that are subsequently used for RNA synthesis or sequencing.
- DNA Sequencing: Another application of the T7 promoter primer is in DNA sequencing, particularly in Sanger sequencing. When the T7 promoter is part of the vector, this primer can be used to initiate sequencing reactions from the T7 promoter region within the plasmid. This helps confirm the integrity of the plasmid and ensures that the gene of interest is cloned correctly with the T7 promoter in the proper orientation. Sequencing with the T7 primer also validates sequence accuracy, ensuring that there are no mutations in the T7 promoter region that could affect transcription efficiency.
