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Bioinformatics Advance Access published online on August 16, 2005
Bioinformatics, doi:10.1093/bioinformatics/bti633
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© The Author (2005). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org
Received April 20, 2005
Revised August 14, 2005
Accepted August 15, 2005

Article

Oligonucleotide properties determination and primer designing: a critical examination of predictions

Sreenivas Chavali 1, Anubha Mahajan 1, Rubina Tabassum 1, Souvik Maiti 2, and Dwaipayan Bharadwaj 3*

1 Functional Genomics Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi-110 007, India
2 Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi-110 007, India
3 Functional Genomics Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi-110 007, India; Proteomics and Structural Biology Unit, Institute of Genomics and Integrative Biology, CSIR, Mall Road, Delhi-110 007, India

* To whom correspondence should be addressed.
Dwaipayan Bharadwaj, E-mail: db{at}igib.res.in


  Abstract

Motivation: Precise prediction of melting temperature (Tm), secondary structures and design of oligonucleotides determine the efficiency and success of experimentation in molecular biology. Availability of a plethora of softwares and the unawareness of the users about their limitations, compromises the accuracy and reliability of the predictions.

Results: Comparative analysis of 56 modules was done for Tm prediction using a large set of oligonucleotide sequences spanning the whole range of GC content and length. Allawi module of the calculator ‘MELTING’, Nearest Neighbor (N.N.) of oligo calculator (McLab), N.N. of Tm Calculation for Oligos (Biomath Calculator- Promega) and HYTHER provided the most precise Tm predictions. A model has also been proposed to calculate optimum annealing temperature integrating the already reported formulations. Secondary structures predictions of oligonucleotides reveal a large number of structures in contrast to the experimental observations. Of the eleven primer designing tools evaluated, Primer 3 and WebPrimer performed the best for the AT rich templates, Exon Primer for AT=GC templates and Primer Design Assistant, Primer3 and Primer Quest for GC rich templates. This study provides optimal choice for application to the user, increasing the success of a variety of experimentations especially those, which have high throughput and complex assay designs.

Supplementary information: The details of the oligonucleotides and of the different modules of Tm prediction considered for the study are provided as supplementary information.


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