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Applied and Environmental Microbiology, July 2009, p. 4747-4752, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00568-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Selection of Enzymes for Terminal Restriction Fragment Length Polymorphism Analysis of Fungal Internally Transcribed Spacer Sequences{triangledown} ,{dagger}

Pablo Alvarado and Jose L. Manjón*

Departamento de Biología Vegetal, Universidad de Alcalá, Ctra. Madrid-Barcelona km 33.6, Alcalá de Henares, 28871 Madrid, Spain

Received 9 March 2009/ Accepted 18 May 2009

Terminal restriction fragment length polymorphism (TRFLP) profiling of the internally transcribed spacer (ITS) ribosomal DNA of unknown fungal communities is currently unsupported by a broad-range enzyme-choosing rationale. An in silico study of terminal fragment size distribution was therefore performed following virtual digestion (by use of a set of commercially available 135 type IIP restriction endonucleases) of all published fungal ITS sequences putatively annealing to primers ITS1 and ITS4. Different diversity measurements were used to rank primer-enzyme pairs according to the richness and evenness that they showed. Top-performing pairs were hierarchically clustered to test for data dependency. The enzyme set composed of MaeII, BfaI, and BstNI returned much better results than randomly chosen enzyme sets in computer simulations and is therefore recommended for in vitro TRFLP profiling of fungal ITSs.

* Corresponding author. Mailing address: Departamento de Biología Vegetal, Universidad de Alcalá, Ctra. Madrid-Barcelona km 33.6, Alcalá de Henares, 28871 Madrid, Spain. Phone: 34 91 885 5071. Fax: 34 91 885 5066. E-mail: josel.manjon{at}uah.es

{triangledown} Published ahead of print on 22 May 2009.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, July 2009, p. 4747-4752, Vol. 75, No. 14
0099-2240/09/$08.00+0     doi:10.1128/AEM.00568-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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