Polymerase Chain Reaction: Types, Applications, Bioinformatics Tools and Guideline for Primer Design

 Polymerase chain reaction (PCR) is a critical and practical technology in various biological and medical sciences fields. Using this technique, millions of copies of a specific pattern can be reproduced in vitro with a minimal amount of DNA. This has made significant advances in molecular biology and medicine. This technique has many applications in biological sciences. PCR primers are an integral part of PCR. Well-designed primers allow the PCR system to work accurately. To create a good PCR primer pair, several factors must be considered, including base pair length, melting and annealing temperature, base pair repeats, etc. Carefully designed PCR primers increase sensitivity and specificity and reduce the effort spent on experimental optimization. The poorly designed primer and the lack of optimization of the reaction conditions probably lead to decreased technical accuracy and false positive or negative detection in the amplification of the target DNA fragments. Bioinformatics has become an essential tool for basic research and applied research in life sciences. Primer designing tools provide optimal and efficient design of primers by automating complex and accurate calculations in the shortest possible time.

 This review article is a content analysis study performed by searching Polymerase Chain Reaction (PCR), PCR type, and primer design for PCR in related articles on Google Scholar, Web of Science, PubMed and Scopus.

 This article aims to provide an almost complete guide for designing efficient and optimal primers for PCR and qPCR analyses by reviewing the PCR method, its types and applications and using the links and web-based services. In this regard, in the first part of the article, various methods, such as standard PCR, reverse transcription PCR (RT-PCR), quantitative PCR (qPCR), combined RT-PCR/qPCR, etc., will be explained with suitable figures. The following summarizes the use of PCR types, emphasizing cereal research. In the end, primer design methods, especially for qPCR, classification and review of existing software and their capabilities, methods and software for testing the features of primers to obtain maximum specificity and PCR efficiency, extracting the gene sequence for PCR primer designing is presented with practical examples.

 A PCR with weak primers can produce little or no product due to non-specific amplification or primer dimer formation. Several online tools were introduced that are used to prepare effective primers for PCR in the field of molecular biology.