EVALUATING THE PHYSICAL CAPTURE METHOD OF TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM

Authors: Blackwood, Christopher; Buyer, Jeffrey

Submitted to: Soil Biology and Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/7/2006
Publication Date: 2/1/2007
Citation: Blackwood, C.B., Buyer, J.S. 2007. Evaluating the physical capture method of terminal restriction fragment length polymorphism. Soil Biology and Biochemistry. 39:590-599.

Interpretive Summary: Molecular genetic methods are used to monitor microbial populations in soils because the soil community is highly complex and most organisms are unable to grow outside their natural habitat. Currently, molecular methods are used which attempt to monitor all taxonomic groups simultaneously, resulting in the ability to detect differences between communities, but not to attribute changes to any particular microbial groups. We developed a modification of a method commonly used to fingerprint community DNA called terminal restriction fragment length polymorphism (T-RFLP). The standard T-RFLP method requires the use of a DNA sequencing instrument to determine the length of each DNA fragment, but there is no way to actually determine the sequence of each fragment. With our modification the length of each DNA fragment can be determined on an ordinary gel in the laboratory, and fragments of interest can be cut out of the gel and sequenced. Sequence information can then be used to determine the taxonomic affiliation of the DNA fragment, and identify the microbial populations that differ between different samples. We demonstrated that results were similar by the standard method and the modified method, and also that sequencing the DNA fragments provides important information that cannot be obtained just by determining the length of the fragment. This methodn may be useful in risk assessment and other studies where it is necessary to determine the impact of a particular treatment, such as introduction of a genetically modified organism or conversion of a farm from conventional to sustainable agriculture, on soil microbial communities.

Technical Abstract: Terminal restriction fragment length polymorphism (T-RFLP) is a popular method of comparative microbial community analysis which is normally accomplished by tagging terminal restriction fragments (T-RFs) with a fluorescent primer. Here we evaluate an alternative method of T-RFLP where T-RFs are physically captured using a biotinylated primer and streptavidin-coated beads. This eliminates one of the primary criticisms of T-RFLP, namely that T-RFs cannot be identified by sequence analysis, and also represents an alternative method for collecting T-RFLP profiles. Microbial communities from forest, agricultural, and turf soils were investigated using several sets of primers specific for different microbial groups. The physical capture method of T-RFLP resulted in very similar profiles to those generated by fluorescent T-RFLP. The relationships among ecosystem types captured by both methods were virtually identical. The total variance in the profiles that was attributed to ecosystem type was approximately equal, or greater, when generated by the physical capture method, depending on the primers used. However, physical capture T-RFLP resolved fewer T-RFs than fluorescent T-RFLP. Direct cloning and sequencing of physical capture T-RFs revealed that most bands were not comprised of sequences related to those in the database that would generate T-RFs of similar size. We therefore suggest that T-RFs should be identified by sequencing, rather than by comparing the sizes of T-RFs to computer digests of database sequences. Physical capture T-RFLP should be a useful tool to identify T-RFs by sequencing, and for laboratories without economical access to sequencing equipment required to perform fluorescent T-RFLP.