Author:
Brodin Johanna,Krishnamoorthy Mohan,Athreya Gayathri,Fischer Will,Hraber Peter,Gleasner Cheryl,Green Lance,Korber Bette,Leitner Thomas
Abstract
Abstract
Background
Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design.
Results
Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations.
Conclusions
PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples.
Publisher
Springer Science and Business Media LLC
Subject
Applied Mathematics,Computer Science Applications,Molecular Biology,Biochemistry,Structural Biology
Reference32 articles.
1. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, Rozen SG: Primer3-new capabilities and interfaces. Nucleic Acids Res. 2012, 40 (15): e115-10.1093/nar/gks596.
2. Rychlik W: OLIGO 7 primer analysis software. Methods Mol Biol. 2007, 402: 35-60. 10.1007/978-1-59745-528-2_2.
3. Vallone PM, Butler JM: AutoDimer: a screening tool for primer-dimer and hairpin structures. Biotechniques. 2004, 37 (2): 226-231.
4. Kalendar R, Lee D, Schulman AH: Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics. 2011, 98 (2): 137-144. 10.1016/j.ygeno.2011.04.009.
5. Giegerich R, Meyer F, Schleiermacher C: GeneFisher-software support for the detection of postulated genes. Proc Int Conf Intell Syst Mol Biol. 1996, 4: 68-77.
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