I. Introduction
Primer sequences are indispensable elements in various molecular biology techniques, with a primary application in the Polymerase Chain Reaction (PCR). These short, single-stranded DNA sequences are meticulously designed to target specific regions of a DNA molecule. They act as the initiation point for DNA synthesis during the amplification process in PCR. The meticulous design of primer sequences entails several critical factors. These include the sequence length, GC content, melting temperature (Tm), and specificity. The interaction between these factors significantly influences the outcome of DNA amplification.
Primer sequences assume a pivotal role in a spectrum of PCR applications, encompassing DNA amplification, gene expression analysis, genotyping, and mutation detection. These sequences are foundational tools in molecular biology research, facilitating genetic investigations in numerous fields.
II. Primer Sequence
In our project, we employed Primer3, a widely recognized primer design tool (https://bioinfo.ut.ee/primer3-0.4.0/). The design of the primer sequences was rooted in the comprehensive information available in the NCBI nucleoside database. Once the primer was designed, it was synthesized using the solid-phase oligonucleotide synthesis method, employing automated DNA synthesizers. The adherence to detailed design guidelines and the precision of the sequence synthesis are pivotal for achieving successful DNA amplification and accurate results in molecular biology experiments.
III. Result
The result by Primer3 using 1-based sequence positions: