In Silico PCR Primer Designing and Validation

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1275)

Abstract

Polymerase chain reaction (PCR) is an enzymatic reaction whose efficiency and sensitivity largely depend on the efficiency of the primers that are used for the amplification of a concerned gene/DNA fragment. Selective amplification of nucleic acid molecules initially present in minute quantities provides a powerful tool for analyzing nucleic acids. In silico method helps in designing primers. There are various programs available for PCR primer design. Here we described designing of primers using web-based tools like “Primer3” and “Web Primer”. For designing the primer, DNA template sequence is required that can be taken from any of the available sequence databases, e.g., RefSeq database. The in silico validation can be carried out using BLAST tool and Gene Runner software, which check their efficiency and specificity. Thereafter, the primers designed in silico can be validated in the wet lab. After that, these validated primers can be synthesized for use in the amplification of concerned gene/DNA fragment.

Key words

Polymerase chain reaction (PCR) Primer3 Web Primer Gene Runner DNA polymerase RAPD DNA isolation Purification Validation 

Notes

Acknowledgements

The authors acknowledge the facilities of the Department of Biotechnology, Ministry of Science and Technology, Government of India, New Delhi (DBT), under the Bioinformatics subcentre in the preparation of this manuscript.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.School of BiotechnologyDevi Ahilya UniversityIndoreIndia

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