Skip to main content

Identification of Homologous Gene Sequences by PCR with Degenerate Primers

  • Protocol
  • First Online:
Molecular Methods for Evolutionary Genetics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 772))


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).

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Subscribe and save

Springer+ Basic
EUR 32.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or Ebook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others


  1. Hall T (2005) BioEdit Sequence Alignment Editor for Windows 95/98/NT/XP. Accessed 2010 Oct 20

  2. 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

    Google Scholar 

  3. 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

    Google Scholar 

  4. 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

    Google Scholar 

  5. 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

    Google Scholar 

  6. Edgar R (2004) MUSCLE: Multiple sequence alignment with high score accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Google Scholar 

  7. Do CB, Mahabhashyam MS, Brudno M et al (2005) ProbCons: Probabilistic consistency-based multiple sequence alignment. Genome Research 15:330–340

    Google Scholar 

  8. Notredame C, Higgins D, Heringa J (200) T-Coffee: a novel algorithm for multiple sequence alignment. J Mol Biol 302:205–217

    Google Scholar 

  9. Marshall OJ (2005) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20: 2471–2472

    Google Scholar 

  10. EMBL, EBI, Wellcome Trust Sanger Institute (2010) Ensembl release 59. Accessed 2010 Oct 20

  11. U.S. National Library of Medicine (2010) Nucleotide home. Accessed 2010 Oct 20

  12. University of California Santa Cruz Genome Bioinformatics (2010) UCSC Genome Browser. Accessed 2010 Oct 20

  13. Sambrook J, Russell DW (2000) Molecular cloning: a laboratory manual. 3 éd. New York: Cold Spring Harbor Laboratory Press

    Google Scholar 

  14. 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

    Google Scholar 

  15. Moura GR, Paredes JA, Santos MA (2010) Development of the genetic code: Insights from a fungal codon reassignment. FEBS Letters 584:334–341

    Google Scholar 

Download references


Our work is supported by an ATIP-AVENIR grant to VO and by a postdoctoral fellowship from the French Foreign Ministry to ML.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Michael Lang .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

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.

Download citation

  • DOI:

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61779-227-4

  • Online ISBN: 978-1-61779-228-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics