Abstract
Degenerate primers are mixtures of similar oligonucleotides that are used in a PCR, so-called degenerate PCR, to amplify unknown DNA sequences, typically coding sequences of genes. Degenerate primers are designed based on sequence data of related and already sequenced gene homologs. This method is useful for identifying new members of a gene family or orthologous genes from different organisms where genomic information is not available. We describe here how to design degenerate primers, set up the PCR (with genomic DNA or cDNA as a template), clone the resulting PCR fragments, and sequence them. Since this method only yields partial coding sequences, complete gene sequences must then be achieved by other approaches such as inverse PCR (see Chapter 16), 5′ RACE, 3′ RACE, or circular RACE (see Chapter 15).
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References
Hall T (2005) BioEdit Sequence Alignment Editor for Windows 95/98/NT/XP. http://www.mbio.ncsu.edu/BioEdit/bioedit.html. Accessed 2010 Oct 20
Gouy M, Guindon S, Gascuel O (2010) SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building. Molecular Biology and Evolution 27:221–224
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22:4673–4680
Subramanian A, Kaufmann M, Morgenstern B (2008) DIALIGN-TX: greedy and progressive approaches for segment-based multiple sequence alignment. Algorithms for Molecular Biology 3:6
Katoh K, Misawa K, Kuma K et al (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059–3066
Edgar R (2004) MUSCLE: Multiple sequence alignment with high score accuracy and high throughput. Nucleic Acids Res 32:1792–1797
Do CB, Mahabhashyam MS, Brudno M et al (2005) ProbCons: Probabilistic consistency-based multiple sequence alignment. Genome Research 15:330–340
Notredame C, Higgins D, Heringa J (200) T-Coffee: a novel algorithm for multiple sequence alignment. J Mol Biol 302:205–217
Marshall OJ (2005) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20: 2471–2472
EMBL, EBI, Wellcome Trust Sanger Institute (2010) Ensembl release 59. http://www.ensembl.org/index.html. Accessed 2010 Oct 20
U.S. National Library of Medicine (2010) Nucleotide home. http://www.ncbi.nlm.nih.gov/nuccore. Accessed 2010 Oct 20
University of California Santa Cruz Genome Bioinformatics (2010) UCSC Genome Browser. http://genome.ucsc.edu/index.html. Accessed 2010 Oct 20
Sambrook J, Russell DW (2000) Molecular cloning: a laboratory manual. 3 éd. New York: Cold Spring Harbor Laboratory Press
Nomenclature Committee of the International Union of Biochemistry (NC-IUB) (1985) Nomenclature for incompletely specified bases in nucleic acid sequences. Biochem J 229: 281–286
Moura GR, Paredes JA, Santos MA (2010) Development of the genetic code: Insights from a fungal codon reassignment. FEBS Letters 584:334–341
Acknowledgments
Our work is supported by an ATIP-AVENIR grant to VO and by a postdoctoral fellowship from the French Foreign Ministry to ML.
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Lang, M., Orgogozo, V. (2012). Identification of Homologous Gene Sequences by PCR with Degenerate Primers. In: Orgogozo, V., Rockman, M. (eds) Molecular Methods for Evolutionary Genetics. Methods in Molecular Biology, vol 772. Humana Press. https://doi.org/10.1007/978-1-61779-228-1_14
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DOI: https://doi.org/10.1007/978-1-61779-228-1_14
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