Primer designing in Primer-BLAST

Abstract

The polymerase chain reaction (PCR) is widely used in different areas. For example, in laboratory diagnostics, PCR is used to detect bacterial and viral pathogens, in the diagnosis of hereditary diseases, and to identify paternity and many other. There are three types of PCR — qualitative, semi-quantitative, and quantitative. The method is based on the ability of DNA polymerase to double the existing DNA strand, thus resulting in multiplication the number of copies of the region of interest. Necessary components of the reaction are DNA or RNA molecules, serving as a template for the of new molecules; polymerase — an enzyme synthesizes new DNA strands; short oligonucleotides – primers that are required for the enzyme work and are specific to the fragment of interest. Currently, there are not many primer designing tools that are easy-to-use and free. One of these tools is the Primer-BLAST online resource, which is integrated with the NCBI database. It is user-friendly and easyto-use effective tool that is integrated with GenBank and RefSeq, and always is up-to-dated. However, even for this tool, there are no detailed instructions for the primers designing. This work is an update for a previously published tutorial and provides a step-by-step guide to find target DNA regions, primers designing, and performing primer quality control. The paper is largely based on the personal experience of the authors and is intended for researchers working with PCR.